Recombinant human/bovine parainfluenza virus 3 (B/HPIV3) expressing a chimeric RSV/BPIV3 F protein and uses thereof

ABSTRACT

Recombinant paramyxoviruses including a viral genome encoding a heterologous gene are provided. In several embodiments, the recombinant paramyxovirus is a recombinant parainfluenza virus, such as a recombinant PIV3 including a viral genome encoding a heterologous respiratory syncytial virus F ectodomain linked to the transmembrane domain and the cytoplasmic tail of the F protein from the PIV3. Nucleic acid molecules including the genome of a recombinant paramyxoviruses are also provided. The recombinant viruses may advantageously be used in vaccine formulations, such as for vaccines against parainfluenza virus and respiratory syncytial virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US2016/014154, filed Jan. 20, 2016, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 62/105,667, filed Jan. 20, 2015. Theprovisional application is incorporated by reference herein in itsentirety.

FIELD

This disclosure relates to recombinant paramyxoviruses that include aviral genome including a heterologous gene encoding an antigen of aheterologous virus. For example, the recombinant paramyxovirus can be arecombinant parainfluenza virus (PIV) that includes a genome including aheterologous gene encoding a respiratory syncytial virus (RSV) fusion(F) protein.

BACKGROUND

Paramyxoviruses are a family of negative-sense single stranded RNAviruses that account for many animal and human deaths worldwide eachyear. The paramyxoviruses include sub-families Paramyxovirinae andPneumovirinae. Respiratory syncytial virus (RSV) is an envelopednon-segmented negative-strand RNA virus in the family Paramyxoviridae,genus Pneumovirinae. It is the most common cause of bronchiolitis andpneumonia among children in their first year of life. RSV also causesrepeated infections including severe lower respiratory tract disease,which may occur at any age, especially among the elderly or those withcompromised cardiac, pulmonary, or immune systems. Passive immunizationcurrently is used to prevent severe illness caused by RSV infection,especially in infants with prematurity, bronchopulmonary dysplasia, orcongenital heart disease. Despite the burden of RSV infection in certainpopulations, development of an effective RSV vaccine remains elusive.

Parainfluenza virus (PIV) is another enveloped non-segmentednegative-strand RNA virus that, like RSV, is in the paramyxovirusfamily. However, PIVs are in subfamily Paramyxovirinae. PIVs includemembers of the genus respirovirus (including PIV1, PIV3, Sendai virus)and rubulavirus (including PIV2, PIV4, PIV5). In addition the members ofgenus avulavirus (including Newcastle disease virus NDV) historicallywere termed PIVs and operationally can be considered the same. The humanparainfluenza viruses (HPIVs, serotypes 1, 2, and 3) are second only toRSV in causing severe respiratory infections in infants and childrenworldwide, with HPIV3 being the most important of the HPIVs in terms ofdisease impact. The HPIV genome is approximately 15.5 kb, including agene order of 3′-N-P-M-F-HN-L. Each gene encoding a separate mRNA thatencodes a major protein: N, nucleoprotein; P, phosphoprotein; M, matrixprotein; F, fusion glycoprotein; HN, hemagglutinin-neuramindaseglycoprotein; L, large polymerase protein. The P gene contains one ormore additional open reading frames (ORFs) encoding accessory proteins.Similar to RSV, development of an effective HPIV vaccine remainselusive.

SUMMARY

Recombinant paramyxoviruses including a viral genome encoding aheterologous gene are provided. In several embodiments, the recombinantparamyxovirus can be a recombinant parainfluenza virus comprising aviral genome comprising a heterologous gene encoding a type I membraneprotein comprising a recombinant RSV F ectodomain linked to acytoplasmic tail (CT), or a transmembrane domain (TM) and a CT, of an Fprotein of the paramyxovirus. The paramyxovirus can be, for example, arecombinant human/bovine parainfluenza virus 3 (B/HPIV3), a recombinanthuman parainfluenza virus 1 (HPIV1), a recombinant human parainfluenzavirus 2 (HPIV2), a recombinant human parainfluenza virus 3 (HPIV3), or arecombinant bovine parainfluenza virus 3 (BPIV3).

Surprisingly, swapping the TM and CT of the heterologous RSV F proteinfor the corresponding TM and CT of the paramyxovirus F protein provideda multi-fold increase in RSV F ectodomain incorporation in the envelopeof recombinant paramyxovirus, and dramatically increased the elicitationof an immune response to the ectodomain when the recombinantparamyxovirus was administered to a subject. Further, the induction ofvirus-neutralizing serum antibodies was dramatically increased both inquantity and in quality. Accordingly, in several embodiments, thedisclosed recombinant paramyxoviruses can be included in immunogeniccompositions for eliciting a bivalent immune response to theparamyxovirus and the heterologous RSV F protein.

The RSV F ectodomain encoded by the heterologous gene can be from ahuman RSV F protein. In several embodiments the RSV F ectodomain caninclude one or more amino acid substitutions (such as the “DS-Cav1”substitutions, S155C, S290C, S190F, and V207L) to stabilize theectodomain in a RSV F prefusion conformation. In additional embodiments,the RSV F ectodomain can include one more amino acid substitutions toincrease ectodomain expression or incorporation in the viral envelope(such as the “HEK” substitutions, K66E and Q101P).

In a non-limiting embodiment, the recombinant paramyxovirus can be arecombinant B/HPIV3 and the RSV F ectodomain is linked to a TM and CTfrom a BPIV3 F protein. In some such embodiments, the RSV F ectodomainlinked to the TM and CT from the BPIV3 F protein comprises the aminoacid sequence set forth as SEQ ID NO: 21, or an amino acid sequence atleast 90% identical to SEQ ID NO: 21.

In several embodiments, the recombinant paramyxovirus is a recombinantPIV comprising a viral genome comprising, from upstream to downstream: aPIV genomic promoter followed by the N, P, M, F, HN, and L genes. Insome such embodiments, the heterologous gene included in the viralgenome can be located between the genomic promoter and the gene encodingthe N protein, or between the genes encoding the N and the P protein.

In additional embodiments, the heterologous gene included in the viralgenome of the recombinant paramyxovirus can be codon-optimized forexpression in human cells. In more embodiments, the recombinantparamyxovirus can be an attenuated virus. In other embodiments, theadded gene and its encoded protein can provide attenuation needed for avaccine candidate.

Immunogenic compositions including the recombinant paramyxovirus arealso provided. The compositions can further include an adjuvant. Methodsof generating an immune response in a subject by administering aneffective amount of a disclosed recombinant paramyxovirus to the subjectare also disclosed. Further provided are isolated nucleic acid moleculesincluding the viral genome of any of the recombinant paramyxovirusesdisclosed herein.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Construction of rB/HPIV3 vectors expressing versions of the RSVF protein containing the non-HEK or HEK amino acid assignments. The FORFs were codon-optimized for human expression using the GeneArt (GA)algorithm. The constructs were called non-HEK/GA-opt and HEK/GA-opt. TheHEK (66E, 101P) and non-HEK (66K, 101Q) amino acid assignments areindicated by asterisks. Other annotations: S, signal sequence; p27, 27kprotein fragment liberated by cleavage-activation; FP, fusion peptide;TM, transmembrane; CT, cytoplasmic tail. The RSV F ORFs were placedunder the control of BPIV3 gene-start and gene-end transcription signalsand inserted into the 2^(nd) genome position between the N and P genesof the B/HPIV3 vector. The rB/HPIV3 vector includes N, P, M, and L genesfrom BPIV3, and F and NH genes from HPIV3. The same vector genomeposition and vector transcription signals were used for all of the otherrB/HPIV3 vectors expressing RSV F protein described in FIGS. 1-35.

FIGS. 2A and 2B. The presence of the HEK assignments in the RSV Fprotein resulted in increased protein expression and a reduction inprotein trimer mobility in polyacrylamide gel electrophoresis comparedto that of non-HEK F protein. Vero cells were infected with vectorsexpressing HEK or non-HEK RSV F (from GA-optimized ORFs, shown inFIG. 1) at an MOI of 10 TCID₅₀ at 32° C. Cell lysates were prepared at48 hours post-infection. Equal amounts of cell lysates were analyzed byelectrophoresis after being boiled and reduced (A) or without beingboiled and reduced (B). Denatured and reduced RSV F monomer was detectedwith a commercially-obtained RSV F-specific mouse monoclonal antibody(A). Native RSV F trimer was detected with polyclonal antibodies raisedin rabbits by repeated immunizations with sucrose purified RSV particles(B).

FIGS. 3A and 3B. Formation of syncytia in Vero cell monolayers infectedwith rB/HPIV3 vectors expressing non-HEK or HEK RSV F protein. Cellswere infected with rB/HPIV3 expressing GA-codon-optimized RSV F (seeFIG. 1) with (A) non-HEK or (B) HEK assignments at an MOI of 10 TCID₅₀at 32° C. Images of the infected cells were acquired at 48 hourspost-infection. Representative syncytia are marked with dashed outline.

FIG. 4. Construction of rB/HPIV3 vectors expressing RSV F ORFs that werecodon-optimized (for human expression) by different algorithms andcontained the HEK assignments. The ORF encoding the RSV F protein withHEK assignments was optimized for human codon usage with the GAalgorithm (HEK/GA-opt, shown in FIG. 1), the DNA2.0 algorithm(HEK/D2-opt), or the GenScript (GS) algorithm (HEK/GS-opt). Thesecodon-optimized ORFs were compared with the non-HEK, non-optimizedversion of the RSV F ORF (Non-HEK/non-opt). These RSV F ORFs wereinserted into the rB/HPIV3 vector in exactly the same position and withthe same vector signals as in FIG. 1.

FIGS. 5A and 5B. Increased in vitro expression of RSV F protein fromrB/HPIV3 vectors due to the HEK assignments and codon optimization.Expression of RSV F in (A) Vero and (B) LLC-MK2 cells was evaluated byWestern blot analysis. Cells were infected at an MOI of 10 TCID₅₀ at 32°C. with the indicated rB/HPIV3 vectors, and cell lysates were harvestedat 48 hours post-infection. Lysates were subjected to gelelectrophoresis under reducing and denaturing conditions and analyzed byWestern blotting. Proteins were visualized by reaction with fluorescentantibodies and detected by infrared imaging. The experiment wasperformed with a total of three wells per virus. A monoclonal antibodyspecific to RSV F detected the uncleaved F₀ precursor and cleaved F₁subunit. RSV F₁ band densities were quantified and normalized to theband density of the Non-HEK/non-opt samples indicated as “1”. Expressionof the HPIV3 HN protein also was determined as an internal control forvector protein expression and to ensure equivalence of MOI andreplication; β-actin was used as the loading control.

FIG. 6. Effects of HEK and codon-optimization of the F ORF on theformation of syncytia in vector-infected Vero cell monolayers. Cellswere mock-infected (mock) or infected with empty rB/HPIV3 vector (emptyB/H3) or with rB/HPIV3 vector expressing the RSV F ORF that was non-HEKand non-optimized (Non-HEK/non-opt) or was HEK and GA-optimized(HEK/GA-opt) or HEK and DNA2.0-optimized (HEK/D2-opt) or HEK andGS-optimized (HEK/GS-opt). Infections were performed at an MOI of 10TCID₅₀ at 32° C. and images were acquired at 48 hours post-infection.Representative syncytia are indicated with dashed outline in some of thepanels.

FIGS. 7A and 7B. Multi-cycle in vitro replication of rB/HPIV3 vectorsexpressing HEK or non-HEK RSV F protein from non-optimized orcodon-optimized ORFs. (A) LLC-MK2 and (B) Vero cells were infected intriplicate at 32° C. at an MOI of 0.01 TCID₅₀ with empty rB/HPIV3 vector(empty B/H3) or vector expressing the RSV F ORF that wasnon-HEK-containing and non-optimized (Non-HEK/non-opt) or wasnon-HEK-containing and GA-optimized (Non-HEK/GA-opt) or wasHEK-containing and GA-optimized (HEK/GA-opt) or was HEK-containing andGS-optimized (HEK/GS-opt). Aliquots of medium supernatant were collectedat 24 h intervals for 6 days and viral titers were determined bylimiting dilution assay on LLC-MK2 cells at 32° C. and reported asTCID₅₀/ml. Mean titers±SEM from three independent experiments are shown.

FIGS. 8A and 8B. Replication in hamsters of rB/HPIV3 vectors expressingHEK or non-HEK RSV F protein from non-optimized or codon-optimized ORFs.Golden Syrian hamsters were infected intranasally (IN) with 10⁵ TCID₅₀of the indicated rB/HPIV3 vectors or 10⁶ PFU of wt RSV (strain A2) in a0.1 ml inoculum. Hamsters were euthanized (n=6 per virus per day) on day3 and 5 post-infection and the (A) nasal turbinates and (B) lungs wereremoved and homogenized and viral titers were determined by limitingdilution on LLC-MK2 (rB/HPIV3 vectors) or Vero (RSV) cells at 32° C.:open and closed circles indicate titers for animals sacrificed on day 3and 5, respectively. Each symbol represents an individual animal, andthe mean titer of each group is indicated by a dashed and a solidhorizontal line for day 3 and 5, respectively. The limit of detection(LOD) was 1.5 log₁₀ TCID₅₀/g of tissue, indicated with a dotted line.The rB/HPIV3 vectors were titrated by limiting dilution assays onLLC-MK2 cells and reported as TCID₅₀/g; RSV was titrated by plaqueassays on Vero cells and reported as PFU/g.

FIG. 9. Serum RSV-neutralizing antibody titers from hamsters infectedwith rB/HPIV3 vectors expressing HEK or non-HEK RSV F protein fromnon-optimized or codon-optimized ORFs. Hamsters (n=6 animals per virus)were inoculated IN with 10⁵ TCID₅₀ of the indicated rB/HPIV3 vectors or10⁶ PFU of wt RSV in a 0.1 ml inoculum. Serum samples were collected at28 days post-immunization, and RSV-neutralizing antibody titers weredetermined by using a 60% plaque reduction neutralization test (PRNT₆₀)performed on Vero cells at 32° C. in the presence of guinea pigcomplement. Each symbol represents an individual animal. The height ofeach bar represents the mean titer of each group. The values of meantiters are shown above the bars. The standard error of the mean is shownby the horizontal lines. The detection limit for the neutralizationassay was 5.3 reciprocal log₂ PRNT₆₀, indicated with a dotted line.

FIGS. 10A and 10B. Protection of immunized hamsters against RSVchallenge. The hamsters (n=6 animals per virus) that had been immunizedas shown in FIG. 9 with the indicated rB/HPIV3 vectors or with wt RSV,were challenged IN on day 31 post-immunization with 10⁶ PFU of wt RSV ina 0.1 ml inoculum. On day 3 post-challenge, hamsters were euthanized and(A) nasal turbinates and (B) lungs were collected. RSV titers in tissuehomogenates were determined by plaque assay in Vero cells. Each symbolrepresents an individual animal and mean viral titers of the groups areshown as horizontal lines. The detection limit of the assay was log₁₀2.7 PFU/g of tissue, indicated as a dashed line.

FIG. 11. Construction of rB/HPIV3 vectors expressing secreted (Ecto),post-fusion, and stabilized pre-fusion forms of the RSV F protein. Eachof these modified proteins contained the HEK assignments and wasexpressed from a GA-optimized (for human expression) ORF. Annotations:S, signal sequence; p27, 27k protein fragment liberated bycleavage-activation; FP, fusion peptide; TM, transmembrane; CT,cytoplasmic tail. The HEK/GA-opt construct expresses full-length RSV F.The ectodomain or “ecto” form consisted of amino acids 1-513 of the RSVF protein; it lacks the CT and TM anchor and would be available forsecretion. The “post-fusion” form was derived from the ectodomain(1-513aa) by the further deletion of the first 10 aa from the N-terminalend of the fusion peptide (FP; 137-146aa) (McLellan et al, 2011, J Virol85:7788-96). “DS” and “DS-Cav1” are two versions of full-length RSV Fprotein stabilized in the pre-fusion form by the S155C/S290C mutations(DS) or by the DS and S190F/V207L (Cav1) mutations (McLellan et al,2013, Science 342:931). The ORFs encoding these various forms of RSV Fwere inserted into the rB/HPIV3 vector at the same position and with thesame vector signals as described in FIGS. 1 and 4.

FIGS. 12A and 12B. Multi-cycle in vitro replication of rB/HPIV3 vectorsexpressing secreted, post-fusion, and stabilized pre-fusion forms of theRSV F protein. (A) LLC-MK2 and (B) Vero cells were infected at an MOI of0.01 TCID₅₀ with empty rB/HPIV3 vector (empty B/H3) or with theindicated constructs: HEK/GA-opt; Ecto; Post-fusion; and DS (see FIG. 11for descriptions). Viral replication during a period of 6 days at 32° C.was determined by collecting medium supernatant samples at 24-hintervals and performing virus titration by limiting dilution on LLC-MK2cells. See FIG. 11 for diagrams of the mutant proteins. The asterisk *indicates that all of these RSV F constructs were HEK and GA-optimized.

FIGS. 13A and 13B. In vitro expression of secreted (Ecto), post-fusion,and stabilized pre-fusion forms of the RSV F protein from rB/HPIV3vectors. Vero cells were infected with the indicated rB/HPIV3 vectors atan MOI of 10 TCID₅₀ or with wt RSV at an MOI of 10 PFU. Infected cellswere incubated at (A) 32° C. or (B) 37° C. for 48 h. (A) Mediumsupernatants and lysates of cells infected with the rB/HPIV3 vectorsexpressing post-fusion, Ecto, or HEK/GA-opt, or with wt RSV, and (B)lysates of cells infected with rB/HPIV3 vectors with non-HEK/non-opt,HEK/GA-opt, DS, or DS-Cav1 forms of RSV F were harvested and analyzedfor RSV F expression by Western blot. The constructs indicated byasterisk * contained the HEK assignments and were GA-optimized.

FIGS. 14A and 14B. Replication in hamsters of rB/HPIV3 vectorsexpressing secreted (Ecto), post-fusion, and stabilized pre-fusion formsof the RSV F protein. Hamsters were infected IN with 10⁵ TCID₅₀ of theindicated rB/HPIV3 vectors or 10⁶ PFU of wt RSV in a 0.1 ml inoculum.Hamsters were euthanized (n=6 per virus per day) on days 3 and 5post-infection and the (A) nasal turbinates and (B) lungs were removedand homogenized and viral titers were determined by limiting dilution onLLC-MK2 cells (rB/HPIV3 vectors) or Vero (RSV) cells at 32° C.: open andclosed circles indicate titers for animals sacrificed on day 3 and 5,respectively. Each symbol represents an individual animal, and the meantiter of each group is indicated by a dashed or solid horizontal linefor day 3 and 5, respectively. Mean values of day 5 titers are shown atthe top. The rB/HPIV3 vectors were titrated by limiting dilution assayson LLC-MK2 cells and reported as TCID₅₀/g; RSV was titrated by plaqueassays on Vero cells and reported as PFU/g. The limit of detection (LOD)is 1.5 log₁₀ TCID₅₀/g of tissue, indicated with a dotted line. Thestatistical significance of difference among peak titers was determinedby Tukey-Kramer test and indicated by asterisks; *, P≤0.05; **, P≤0.01;or ***, P≤0.001. The constructs indicated by asterisk * contained theHEK assignments and were GA-optimized for human expression.

FIGS. 15A and 15B. Serum RSV-neutralizing antibody titers from hamstersinfected with rB/HPIV3 vectors expressing secreted (Ecto), post-fusion,and stabilized pre-fusion forms of the RSV F protein. Hamsters (n=6animals per virus) were inoculated IN with 10⁵ TCID₅₀ of the indicatedrB/HPIV3 vectors or 10⁶ PFU of wt RSV in a 0.1 ml inoculum. Serumsamples were collected at 28 days post-immunization, andRSV-neutralizing antibody titers were determined by a 60% plaquereduction neutralization test (PRNT₆₀) performed on Vero cells at 32° C.(A) with and (B) without added guinea pig complement. The height of eachbar represents the mean titer. The values of mean titers are shown abovethe bars. The standard error of the mean is shown by the horizontallines. The detection limit for the neutralization assay is indicatedwith a dotted line. ND means neutralization titer is below the detectionlimit. The statistical significance of difference among groups wasdetermined by Tukey-Kramer test and indicated by asterisks; *, P≤0.05;**, P≤0.01; or ***, P≤0.001; or ns, P>0.05.

FIGS. 16A and 16B. Protection of immunized hamsters against RSVchallenge. The hamsters (n=6 animals per virus) that had been immunizedas shown in FIG. 15 were challenged IN on day 31 post-immunization with10⁶ PFU of wt RSV in a 0.1 ml inoculum. On day 3 post-challenge,hamsters were euthanized and (A) nasal turbinates and (B) lungs werecollected. RSV titers in tissue homogenates were determined by plaqueassay in Vero cells at 32° C. Each symbol represents an individualanimal and mean viral titers of the groups are shown as horizontallines. The detection limit of the assay was log₁₀ 2.7 PFU/g of tissue,indicated as a dotted line.

FIGS. 17A and 17B. Construction of rB/HPIV3 vectors expressing versionsof RSV F protein engineered in an attempt to increase incorporation intothe vector particle. (A) Structures of F proteins. (B) Sequences of thecytoplasmic tails (CT), transmembrane (TM) domains, and adjoiningregions of the ectodomains of the RSV F protein (amino acid assignmentsin black) and BPIV3 F protein (boldface), with amino acid sequencepositions indicated. Each of these modified proteins contained the HEKassignments and was expressed from a GA-optimized ORF. The HEK/GA-optconstruct expressed full-length RSV F protein. “B3CT” has the CT of RSVF protein (amino acid sequence positions 551-574) replaced by the CT ofBPIV3 F protein (positions 515-540, boldface). “B3TMCT” has both the TMand CT of RSV F protein (positions 530-574) replaced by the TM and CT ofBPIV3 F protein (positions 494-540, boldface). “DS/B3CT”, “DS/B3TMCT”,“DS-Cav1/B3CT”, and “DS-Cav1/B3TMCT” are versions of B3CT and B3TMCTcontaining the DS or DS-Cav1 mutations designed to stabilize thepre-fusion conformation. The ORFs encoding these various forms of RSV Fprotein were inserted into the rB/HPIV3 vector at the same position andwith the same vector signals as described in FIGS. 1, 4, and 11. Thesequences shown are as follows: RSV(A2)F (SEQ ID NO: 1, residues510-574), BPIV3 F (SEQ ID NO: 151), B3CT (SEQ ID NO: 14, residues510-576), B3TMCT (SEQ ID NO: 12, residues 510-576).

FIGS. 18A and 18B. Incorporation into the rB/HPIV3 vector particle ofB3CT and B3TMCT versions of the RSV F protein. LLC-MK2 cells wereinfected with the indicated rB/HPIV3 vectors at an MOI of 0.01 TCID₅₀ at32° C. The medium supernatants were harvested 6-7 days post-infection,clarified by low speed centrifugation, and subjected to centrifugationon 10%-30% sucrose gradients to obtain partially-purified vectorparticles. Additional Vero cells were infected with wt RSV at an MOI of0.01 PFU and processed in the same way. The protein concentrations ofthe sucrose-purified preparations were determined by a standardcommercial kit. (A) Western blot evaluation of the packaging efficiencyof the RSV F protein into the rB/HPIV3 particles. To compare therelative amounts of RSV F in the particles, 0.5 μg of sucrose-purifiedparticles were lysed, denatured, reduced and subjected to Western blotanalyses. The HPIV3 HN and BPIV3 N proteins of the vector particle werequantified for comparison. (B) The packaging efficiency of each form ofRSV F into its respective vector particle was calculated by normalizingits band density against that of the BPIV3 N protein. The order of thelanes is the same as in part A. The packaging efficiencies of variousforms of RSV F are shown relative to the native F protein set at “1”.The packaging efficiency of the B3CT and B3TMCT forms of RSV F into thevector particle was judged to be similar to that of RSV F into the RSVparticle because the amount of modified RSV F protein per 0.5 μg ofvector particles (lanes 3, 4, 6, 7) was similar to the amount of nativeRSV F protein per 0.5 μg of RSV particles (lane 5). The constructsindicated by asterisk * contained the HEK assignments and wereGA-codon-optimized for human expression.

FIGS. 19A-19F. Visualization of the incorporation of B3CT and B3TMCTversions of the RSV F protein into rB/HPIV3 particles by transmissionelectron microscopy (TEM). Sucrose purified viruses were labeled with anRSV F-specific murine monoclonal antibody and mouse-IgG-specific secondantibodies that were labeled with 6 nm gold particles. Virions and goldparticles were visualized with TEM. Representative images of (A) RSV,(B) empty rB/HPIV3 vector (empty B/H3), (C) vector expressingHEK/GA-opt, (D) vector expressing B3CT, (E) vector expressing B3TMCT,and (F) vector expressing DS/B3TMCT are shown. Arrows point to sporadicgold particles in HEK/GA-opt virions (C). Substantially greater amountsof gold particles associated with the vector particles are evident in D,E, and F.

FIGS. 20A and 20B. Multi-cycle in vitro replication of rB/HPIV3 vectorsexpressing B3CT and B3TMCT versions of the RSV F protein. (A) LLC-MK2and (B) Vero cells were infected at 32° C. with an MOI of 0.01 TCID₅₀with empty rB/HPIV3 vector (empty B/H3) or vector expressing HEK/GA-opt,or B3CT (upper panels), or B3TMCT (upper panels), or DS/B3CT (lowerpanels) or DS/B3TMCT (lower panels). Aliquots of medium supernatant werecollected at 24 h intervals for 6 days and viral titers were determinedby limiting dilution assay on LLC-MK2 cells at 32° C. and reported asTCID₅₀/ml. The constructs indicated by asterisk * contained the HEKassignments and were GA-codon-optimized for human expression.Multiplicity of infection in the assays was 0.01.

FIGS. 21A and 21B. In vitro expression of B3CT and B3TMCT versions ofthe RSV F protein with or without the DS or DS-Cav1 mutations thatstabilize the pre-fusion form of RSV F protein. Expression of (A) B3CTand B3TMCT; and (B) DS and DS-Cav1 in combination with B3CT and B3TMCT.Vero cells were infected with the indicated rB/HPIV3 vectors at an MOIof 10 TCID₅₀, or with RSV at an MOI of 10 PFU. Infected cells wereincubated at (A) 32° C. or (B) 37° C. for 48 h. Cell lysates wereanalyzed for RSV F expression by Western blot. HPIV3 HN protein was usedas a control to show equivalence of vector replication; GAPDH was usedas loading control. The constructs indicated by asterisk * contained theHEK assignments and were GA-codon-optimized for human expression.

FIG. 22. Formation of syncytia in Vero cell monolayers infected withrB/HPIV3 vectors expressing the B3CT or B3TMCT version of the RSV Fprotein with or without the DS mutations that stabilize the pre-fusionform of RSV F protein. Vero cells were infected at an MOI of 10 TCID₅₀with rB/HPIV3 vectors expressing the indicated versions of RSV F proteinand incubated at 32° C. Images were acquired at 48 h post-infection. Theconstructs indicated by asterisk * contained the HEK assignments andwere GA-codon-optimized for human expression.

FIGS. 23A and 23B. Replication in hamsters of rB/HPIV3 vectorsexpressing the B3CT or B3TMCT version of the RSV F protein with orwithout the DS mutations that stabilize the pre-fusion form of RSV Fprotein. Hamsters were infected IN with 10⁵ TCID₅₀ of rB/HPIV3 vectorsor 10⁶ PFU of wt RSV in a 0.1 ml inoculum. Hamsters were euthanized (6per virus per day) on day 3 and 5 post-infection and the (A) nasalturbinates and (B) lungs were removed and homogenized and viral titerswere determined by limiting dilution on LLC-MK2 (rB/HPIV3 vectors) orVero (RSV) cells at 32° C.: open and closed circles indicate titers foranimals sacrificed on day 3 and 5, respectively. Each symbol representsan individual animal, and the mean titer of each group is indicated by adashed or solid horizontal line for day 3 and 5, respectively. Meanvalues of day 5 titers are shown at the top. The rB/HPIV3 vectors weretitrated by limiting dilution assays on LLC-MK2 cells and reported asTCID₅₀/g; RSV was titrated by plaque assays on Vero cells and reportedas PFU/g. The limit of detection (LOD) is 1.5 log₁₀ TCID₅₀/g of tissue,indicated with a dotted line. The statistical significance of differenceamong peak titers was determined by Tukey-Kramer test and indicated byasterisks (*, P≤0.05; **, P≤0.01; or ***, P≤0.001). The constructsindicated by asterisk * along the x-axis contained the HEK assignmentsand were GA-codon-optimized for human expression. Constructs containingthe DS-Cav1 modification were not examined because they were notavailable at the time of this experiment.

FIGS. 24A and 24B. Serum RSV-neutralizing antibody titers from hamstersinfected with rB/HPIV3 vectors expressing the B3CT or B3TMCT version ofthe RSV F protein with or without the DS mutations that stabilize thepre-fusion form of RSV F protein. Hamsters (n=6 animals per virus) wereinoculated IN with 10⁵ TCID₅₀ of the indicated rB/HPIV3 vectors or 10⁶PFU of wt RSV in a 0.1 ml inoculum. Serum samples were collected at 28days post-immunization, and antibody titers were determined by a 60%plaque reduction neutralization test (PRNT₆₀) with (A) or without (B)added guinea pig complement. The height of each bar represents the meantiter shown along with the SEM. The values of mean titers are shownabove the bars. The detection limit for the neutralization assay isindicated with a dotted line. The statistical significance of differencein the mean titers was determined by Tukey-Kramer test and indicated byasterisks (*, P≤0.05; **, P≤0.01; ns, P≥0.05). ND, neutralization titerwas below the detection limit. The constructs indicated by asterisk *along the x-axis contained the HEK assignments and wereGA-codon-optimized for human expression.

FIGS. 25A and 25B. Protection of immunized hamsters against RSVchallenge. The hamsters (n=6 animals per virus) that had been immunizedas shown in FIG. 24 were challenged IN on day 31 post-immunization with10⁶ PFU of wt RSV in a 0.1 ml inoculum. On day 3 post-challenge,hamsters were euthanized and (A) nasal turbinates and (B) lungs werecollected. RSV titers in tissue homogenates were determined by plaqueassay in Vero cells at 32° C. Each symbol represents an individualanimal and mean viral titers of the groups are shown as horizontallines. The detection limit of the assay was log₁₀ 2.7 PFU/g of tissue,indicated as a dotted line.

FIG. 26. Stability of expression of RSV F by rB/HPIV3 vectors duringreplication in hamsters. The percentage of recovered vector expressingRSV F in the nasal turbinates and lungs at day 3 and 5 post-immunizationwas determined by double-staining plaque assay of vector recovereddirectly from the tissue homogenates. The results are expressed for theindividual animals. The percentages of rB/HPIV3 expressing RSV F proteinin the tested specimens are indicated. Specimens with 100% expression ofRSV F protein were colored in yellow; those with 90-99% expression ofRSV F were colored in green; those with 80-89% expression of RSV F werecolored in orange; those with less than 79% expression of RSV F werecolored in red. Specimens that did not generate plaques due to low titerwere marked as “NA”. If the total number of the plaques developed with asample was less than 10, the number of plaques was recorded as “p=X” (Xequals to the number of plaques) in the bracket.

FIG. 27. Temperature sensitivity phenotypes of B/HPIV3 vectors. Theindicated vectors were evaluated for the ability to form plaques onLLC-MK2 cells at the indicated temperatures. Reduction in plaqueformation of ≥100-fold is indicative of temperature sensitivity. Thelowest such restrictive temperature for each virus is indicated in bold,underlining, and is called the shut-off temperature.

FIG. 28. rB/HPIV3 constructs that were evaluated for attenuation andimmunogenicity in non-human primates (Rhesus macaques). Rhesus macaqueswere infected by the combined IN and intratracheal routes with 10⁶TCID₅₀ per site of the following constructs: Non-HEK/non-opt;HEK/GA-opt/DS; and HEK/GA-opt/DS/B3TMCT in groups of five, five and fouranimals, respectively.

FIGS. 29A and 29B. Replication of rB/HPIV3 vectors in rhesus macaques.Rhesus macaques were infected with the indicated rB/HPIV3 vectors asdescribed in FIG. 28. Vector replication in the respiratory tract wasassessed by collecting (A) nasopharyngeal swabs and (B) tracheal lavageson the indicated days and determining the viral titers by limitingdilution assay. Limit of detection is 1.2 log₁₀ TCID₅₀/mL shown asdotted line.

FIG. 30. Serum HPIV3-neutralizing antibody titers induced by rB/HPIV3vectors. Monkey sera were collected at 0, 14, 21, 28, 35 and 56 dayspost-immunization and HPIV3-neutralizing antibody titers were determinedby a 60% plaque reduction neutralization test (PRNT₆₀) in the presenceof added guinea pig complement. The detection limit for theneutralization assay is indicated with a dotted line. The day of RSVchallenge is indicated.

FIGS. 31 and 32. Serum RSV-neutralizing antibody titers induced byrB/HPIV3 vectors. Monkey sera were collected at 0, 14, 21, 28, 35 and 56days post-immunization. (FIG. 31) RSV neutralizing antibody titers atall time points were determined by a 60% plaque reduction neutralizationtest (PRNT₆₀) in the presence of added guinea pig complement. (FIG. 32)RSV neutralizing antibody titers at day 28 post-immunization weredetermined by a 60% plaque reduction neutralization test (PRNT₆₀) in theabsence of added complement. The detection limit for the neutralizationassay is indicated with a dotted line. The statistical significance ofdifference in mean titers was determined by Tukey-Kramer test andindicated by asterisks (**, P≤0.01; ***, P≤0.001). The day of RSVchallenge is indicated.

FIG. 33. Stability of expression of RSV F by rB/HPIV3 vectors duringreplication in rhesus macaques. The percentage of recovered vectorexpressing RSV F in nasal pharyngeal swabs from day 4, 5 and 6post-immunization was determined by double-staining plaque assay. Thepercentages of rB/HPIV3 expressing RSV F in the tested specimens areindicated. Specimens with 100% of viruses expressing RSV F were coloredin yellow; those with 99-90% of viruses expressing RSV F were colored ingreen; those that did not generate plaques due to low titer were markedas “NA”.

FIG. 34. Construction of an rB/HPIV3 vector expressing a secretedversion of HEK/GS-opt/DS-Cav1 RSV F protein that contains a C-terminal“foldon” sequence. RSV F protein containing the HEK assignments andexpressed from a GS-codon-optimized (for human expression) ORF withDS-Cav1 mutations was engineered to contain the N-terminal 513 aminoacids of the F protein (i.e., lacking the TM and CT domains), fused tothe indicated 4-amino acid linker and the indicated 27-amino acid foldonsequence from T4 phage (SEQ ID NO: 132, see Efimov et al. 1994, J MolBiol 242:470-486; Miroshnikov et al 1998 Protein Eng. 11:329-332). TheORF was inserted into the rB/HPIV3 vector at the same position and withthe same vector signals as described in FIGS. 1, 4, 11, and 17.

FIG. 35. Summary of exemplary rB/HPIV3 vectors expressing RSV F,annotated to indicate constructs that have been evaluated in twodifferent studies in hamsters and two different studies in rhesusmonkeys in Example 1.

FIG. 36. Construction of antigenomic cDNAs of the HPIV1 C^(D170) andL^(Y942A) mutants containing the RSV F gene insert at the first (F1),second (F2), or third (F3) genome positions. The rHPIV1 backbones usedfor RSV F expression contained either of the two attenuating mutations:namely the C^(D170) mutation (indicated by *) in the P/C gene or theL^(Y942A) mutation (indicated by •) in L gene. For the HPIV1-F1constructs, the RSV F gene was inserted at the first genome positionbefore the HPIV1 N gene at the MluI site located in the upstreamnon-translated region of the N gene. In case of HPIV1-F2, the RSV F genewas inserted between the HPIV1 N and P genes at the AscI site located inthe upstream non-translated region of the P gene. For the HPIV1-F3, theRSV F gene was cloned between the HPIV1 P and M genes at the NotI sitesituated in the downstream non-translated region of the P gene. For allconstructs, the RSV F ORF was codon optimized for human expression andcontained HEK amino acid assignments. A copy of the N gene-end (GE),intergenic (IG) CTT triplet, and P gene-start (GS) sequence was addedfollowing (F1, F2) or before (F3) the RSV F insert so that it was underthe control of a set of HPIV1 transcription signals. The sequences ofSEQ ID NOs: 138-140 are shown flanking the RSV F insert under HPIV1-F1;the sequences of SEQ ID NOs: 141-143 are shown flanking the RSV F insertunder HPIV1-F2, and the sequences of SEQ ID NOs: 144-145 are shownflanking the RSV F insert under HPIV1-F3.

FIGS. 37A-37D. Multistep replication of HPIV1/RSV-F viruses in Vero (37Aand 37C) and LLC-MK2 (37B and 37D) cells. Triplicate wells of cellmonolayers in 6-well plates were infected at an MOI of 0.01 TCID₅₀ withHPIV1 C^(Δ170) (A and B) or L^(Y942A) (C and D) viruses expressing RSV F(F1, F2, or F3), in parallel with wt HPIV1, HPIV1 L^(Y942A), and HPIV1C^(Δ170). Cultures were incubated at 32° C. Aliquots of cell culturemedium were collected at 24 h intervals and virus titers (log₁₀TCID₅₀/ml ) were determined by serial dilution on LLC-MK2 cells andhemadsorption assay at 32° C. Mean titers with standard errors of themean (SEM) are shown. The statistical significance of difference betweenthe titer of each virus versus wt HPIV1 for day 2 post-infection wasdetermined using the one-way ANOVA with Tukey's multiple comparisonstest and is indicated by asterisks as follows: *, p≤0.05; **, p≤0.01;***, p≤0.001; ****, p<0.0001.

FIGS. 38A-38C. Analysis of the RSV F and HPIV1 vector protein expressionby Western blot. Vero cells were infected with the indicated viruses atan MOI of 5. At 48 h post-infection cells were lysed with SDS samplebuffer. All samples were denatured, reduced and subjected to SDS-PAGEand Western blot. Proteins were transferred onto PVDF membranes andprobed with either RSV F-specific mouse monoclonal antibody or HPIV1 N-,P-, HN-, or F-specific polyclonal antibodies that had been raised byimmunizing rabbits separately with synthetic peptides representing therespective proteins. (A) Bound antibodies were visualized usingcorresponding anti-mouse (IRDye 680LT) and anti-rabbit (IRDye 800CW)antibodies conjugated with infra-red dye. Images were acquired byscanning the blots using the Odyssey infrared imaging system. The imagesshown are from a single experiment that is representative of threeindependent experiments. (B and C) The intensity of protein bands forthe rHPIV1 C^(Δ170) (B) and the rHPIV1 L^(Y942A) (C) constructs wasquantified for three independent experiments and expression is shownrelative to the F3 virus set at 1.0. Plots show data as mean±SEM fromthree independent experiments that were analyzed by one-way ANOVA withDunnett multiple comparisons test using 95% confidence interval.Expression of the HPIV1 proteins by the F1, F2 and F3 viruses wasstatistically compared with that of their corresponding empty vectorbackbone. *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 39A-39I. Formation of cytopathic effects and syncytia on LLC-MK2cell monolayers infected with the rHPIV1 vectors expressing RSV F. MK2cells were infected at an MOI of 0.01 TCID₅₀, incubated for 5 days andimages were acquired at 40× magnification using phase contrast with alight microscope. Photomicrographs of (A) rHPIV1 C^(Δ170)-F1; (B) rHPIV1C^(Δ170)-F2; (C) rHPIV1 C^(Δ170)-F3; (D) rHPIV1 C^(Δ170); (E) rHPIV1L^(Y942A)-F1; (F) rHPIV1 L^(Y942A)-F2; (G) rHPIV1 L^(Y942A)-F3; (H)rHPIV1 L^(Y942A); and (I) wt HPIV1 are shown.

FIGS. 40A and 40B. Replication of RSV F expressing HPIV1 vectors in thenasal turbinates (40A) and lungs (40B) of hamsters. Hamsters wereinoculated intra-nasally with 10⁵ TCID₅₀ of the wt HPIV1, rHPIV1C^(D176) or rHPIV1 L^(Y942A) empty vectors, rHPIV1 C^(D170) or rHPIV1L^(Y942A) expressing RSV F from three genome positions (F1, F2, or F3),rHPIV1-C^(R84G)C^(D170)HN^(553A)L^(Y942A)(a previously-described HPIV1vaccine candidate (Bartlett et al 2007 Virol J 4:6)), or therB/HPIV3-F2, a chimeric bovine/human PIV3 expressing RSV F from the2^(nd) position (also known as HEK/GA-opt, see FIG. 1). Virus titerswere determined in LLC-MK2 cells by hemadsorption assay and reported asLog₁₀ TCID₅₀/g of tissue. Titers for individual animals (6 per group)are shown for day 3 (Δ) and day 5 (•), each symbol representing anindividual animal. The mean values are shown for each group in boldfacefor day 3 and in italicized type for day 5. The limit of detection (LOD)was 1.5 log₁₀ TCID₅₀/ml, indicated with a dotted line across the bottomof each graph. The statistical significance of the difference betweeneach virus versus wt HPIV1 (red asterisks) or versus rB/HPIV3-F2 (bar atthe top) was determined by One-way ANOVA at 95% confidence intervalusing Tukey's multiple comparisons test for day 3 and day 5 p.i. *,p≤0.05; ***, p≤0.001; ****, p≤0.0001; or ns, not significant.

FIGS. 41A and 41B. Protection against wt RSV challenge virus replicationin the nasal turbinates (41A) and lungs (41B) of the immunized hamsters.Hamsters (n=6) in each group were challenged intranasally with 10⁶ PFUof wt RSV A2 at 30 days post-immunization. Nasal turbinates and lungswere collected from euthanized animals on day 3 post-challenge, virustiters were determined for each sample by RSV specific plaque assay onVero cells and reported as Log₁₀ PFU/g of tissue. Mean value for eachgroup is shown in bold face number and by a horizontal bar. Statisticalsignificance of difference among viruses was determined by one-way ANOVAat 95% confidence interval using Tukey's multiple comparisons test andis indicated by *, p<0.05; **, p<0.01; ****p<0.0001; or ns, notsignificant.

FIG. 42 shows a table illustrating the attenuating mutations introducedin the HPIV1 backbone in the P/C or the L ORF. Nucleotide changes(deletion or substitution) in the wt sequence are underlined.

FIG. 43 shows a table illustrating temperature sensitivity ofrecombinant viruses on LLC-MK2 cell monolayers. For temperaturesensitivity, the underlined values in boldface indicate the virusshut-off temperature indicating a temperature sensitive phenotypedefined as the lowest restrictive temperature at which the mean log₁₀reduction in virus titer at a given temperature vs. 32° C. was 2.0 log₁₀or greater than that of the wt rHPIV1 at the same two temperatures. Formonolayers, serial dilutions of each of the indicated viruses on LLC-MK2cells were incubated at various temperatures for 7 days. Virus titerswere determined by hemadsorption with guinea pig erythrocytes andreported as Log₁₀ TCID₅₀/ml with a detection limit of 1.2.

FIG. 44 shows a table illustrating the percentage of virus populationexpressing RSV F after in vivo replication. The percentage of viruspopulation expressing RSV F after in vivo replication (stability) wasdetermined by an immunofluorescent double-staining plaque assay. Verocells were infected with serially diluted tissue homogenates of thenasal turbinates or lungs of infected hamsters (n=6 per virus) collectedon day 3 and 5 p.i. (total 144 samples) and incubated for 6 days undermethylcellulose overlay. Virus plaques were stained with mousemonoclonal anti-RSV F and goat polyclonal anti-HPIV1 specific antibodiesfollowed by detection with the corresponding infrared dye conjugatedsecondary antibodies. Percentage of plaques expressing both RSV F andHPIV1 antigens are shown. The stability of HPIV1 C^(D170)-F1, -F2, andF3 for lung samples and that for HPIV1 L^(Y942A)-F1, -F2, and F3 in theURT and lungs could not be tested due to their lack of replication inthese tissues. Numbers in parenthesis indicate the RSV F expressionstatus for the number of hamsters of the total 6 hamsters per virus. ND,no plaques were detected.

FIG. 45 shows a table listing results indicating that immunization ofhamsters with rHPIV1 expressing RSV F induces serum neutralizingantibodies against RSV. Groups of six-week old hamsters (n=6) wereintranasally immunized with 10⁵ TCID₅₀ of each indicated virus in 0.1 mlinoculum. Serum samples were collected prior to immunization and at 28days post immunization. Antibody titers against RSV and HPIV1 weredetermined by using a 60% plaque reduction neutralization test (PRNT₆₀)using green fluorescent protein (GFP)- or enhanced GFP (eGFP) expressingviruses (rRSV-eGFPM or HPIV1-GFP), and neutralizing antibody titers werepresented as mean reciprocal log₂±SE. Based on the initial serumdilutions used in the assay, the PRNT₆₀ assay has a titer detectionlimit of 3.3 and 1.0 reciprocal log₂ PRNT₆₀ for RSV and HPIV1,respectively. Statistical significance of difference among the groupsfor RSV antibody titers was determined by one-way ANOVA with Tukey'smultiple comparisons test (p<0.05) and that for HPIV1 antibody titerswas determined by Unpaired t-test. Mean neutralizing antibody titerswere categorized into groups (indicated in parenthesis as A, B, C, andD). Mean antibody titers of treatment groups with different letters arestatistically different from each other; titers shown with two lettersare not statistically different from those indicated with either letter.

FIGS. 46 and 47. Multi-cycle in vitro replication of rB/HPIV3 vectorsexpressing GA-optimized (GA-opt) prefusion form of RSV F with DS-Cav1mutations. (FIG. 46) Vero and (FIG. 47) LLC-MK2 cells were infected intriplicate at 32° C. at an MOI of 0.01 TCID₅₀ with empty rB/HPIV3 vector(empty B/H3) or vector expressing the RSV F ORF that was HEK-containing,GA-opt, and containing the DS-Cav1 prefusion stabilizing mutations(HEK/GA-opt/DS-Cav1) or was HEK-containing, GA-opt, and containing theDS-Cav1 mutations and BPIV3-specific TM and CT domains as potentialpackaging signals (HEK/GA-opt/DS-Cav1/B3TMCT). Aliquots of mediumsupernatants were collected at 24 h intervals for 6 days and viraltiters were determined by limiting dilution assay on LLC-MK2 cells at32° C. and reported as TCID₅₀/ml. Mean titers±SEM from three independentexperiments are shown.

FIGS. 48A and 48B. Multi-cycle in vitro replication of rB/HPIV3 vectorsexpressing GS-optimized (GS-opt) RSV F with different modifications. (A)Vero and (B) LLC-MK2 cells were infected in triplicate at 32° C. at anMOI of 0.01 TCID₅₀ with empty rB/HPIV3 vector (empty B/H3) or vectorexpressing the RSV F ORF that was HEK-containing and GS-opt RSV F(HEK/GS-opt), or was HEK-containing, GS-opt and bearing DS-Cav1prefusion stabilizing mutations (HEK/GS-opt/DS-Cav1), or wasHEK-containing, GS-opt, and bearing the DS-Cav1 mutations andBPIV3-specific TM and CT domains (HEK/GS-opt/DS-Cav1/B3TMCT), or was atruncated RSV F with amino acids from 1 to 513 that was fused to afour-amino acid linker and 27-amino acid oligomerization sequence fromT4 phage, which was HEK-containing, GS-opt and bearing DS-Cav1 mutations(HEK/GS-opt/DS-Cav1/(1-513)Foldon). Aliquots of medium supernatants werecollected at 24 h intervals for 6 days and viral titers were determinedby limiting dilution assay on LLC-MK2 cells at 32° C. and reported asTCID₅₀/ml. Mean titers±SEM from three independent experiments are shown.

FIGS. 49A-49D. Comparison of multi-cycle in vitro replication ofrB/HPIV3 vectors expressing GS-opt and GA-opt RSV F. FIGS. 49A and 49B:(A) Vero and (B) LLC-MK2 cells were infected in triplicate at 32° C. atan MOI of 0.01 TCID₅₀ with empty rB/HPIV3 vector (empty B/H3) or vectorexpressing RSV F ORF that was HEK-containing, GS-opt, and bearing theDS-Cav1 mutations (HEK/GS-opt/DS-Cav1), or was HEK-containing, GA-opt,and bearing the DS-Cav1 mutations (HEK/GA-opt/DS-Cav1). FIGS. 49C and49D: (C) Vero and (D) LLC-MK2 cells were infected at 32° C. at an MOI of0.01 TCID₅₀ with empty rB/HPIV3 vector (empty B/H3) or vector expressingRSV F ORF that was HEK-containing, GS-opt, and bearing the DS-Cav1 andB3TMCT modifications (HEK/GS-opt/DS-Cav1/B3TMCT), or was HEK-containing,GA-opt, and contained the DS-Cav1 and B3TMCT modifications(HEK/GA-opt/DS-Cav1/B3TMCT). Aliquots of medium supernatant werecollected at 24 h intervals for 6 days and viral titers were determinedby limiting dilution assay on LLC-MK2 cells at 32° C. and reported asTCID₅₀/ml. Mean titers±SEM from three independent experiments are shown.

FIGS. 50A-50C. Expression of various modified forms of RSV F by rB/HPIV3vectors in cell culture. (A) Vero and (B, C) LLC-MK2 cells were infectedwith empty rB/HPIV3 vector (lane 1), or rB/HPIV3 vector expressing theindicated modified forms of RSV F (lanes 2-5 and 8), or wt RSV (wt RSV,lane 6) at MOI of 3 PFU/cell, or uninfected (mock, lane 7). InfectedVero (A) and LLC-MK2 (B) cells were incubated at 32° C., and LLC-MK2 (C)cells were incubated at 37° C. Cell lysates and medium supernatant ofVero cells were collected at 48 hpi and were subjected to Western blotanalysis for the expression of RSV F, which was detected as cleaved F₁and/or un-cleaved F₀ forms. BPIV3 N was used as an internal control forthe expression of vector protein; GAPDH was used as a loading control.

FIGS. 51A and 51B. Replication of rB/HPIV3 vectors in the upper andlower respiratory tract of hamsters. Hamsters were infected IN with 10⁵TCID₅₀ of the indicated rB/HPIV3 vectors or 10⁶ PFU of wt RSV in a 0.1ml inoculum. Hamsters were euthanized (n=6 per virus per day) on days 4and 5 post-infection and the (A) nasal turbinates and (B) lungs wereremoved and homogenized, and viral titers were determined by limitingdilution on LLC-MK2 cells at 32° C. and reported as TCID₅₀/g (rB/HPIV3vectors) or were determined by plaque assays on Vero cells at 32° C. andreported as PFU/g (wt RSV). The limit of detection (LOD) is 1.5 log₁₀TCID₅₀/g of tissue, indicated with a dotted line. Open and closedcircles indicate titers for individual animals sacrificed on day 4 and5, respectively. The mean titers of each group are indicated by a dashedand solid horizontal line for day 4 and 5, respectively. The values ofthe mean titers on day 4 and 5 are shown at the top. The mean viraltiters on day 5 were assigned to different groups using the Tukey-Kramertest: mean titers with different letters are statistically different(p<0.05), whereas titers indicated with two letters are notsignificantly different than those indicated with either letter.

FIGS. 52 and 53. Serum RSV-neutralizing antibody titers from hamstersinfected with the indicated rB/HPIV3 vectors expressing the GA-opt orGS-opt RSV F protein with or without the DS or DS-Cav1 or B3TMCTmodifications. Hamsters (n=6 animals per virus) were inoculated IN with10⁵ TCID₅₀ of the indicated rB/HPIV3 vectors or 10⁶ PFU of wt RSV in a0.1 ml inoculum. Serum samples were collected at 28 dayspost-immunization, and antibody titers were determined by a 60% plaquereduction neutralization test (PRNT₆₀) with (FIG. 52) or without (FIG.53) added guinea pig complement. The height of each bar represents themean titer shown along with the SEM. The values of mean titers are shownabove the bars. The pairwise student t-test was used to evaluate thestatistical significance of differences between values: in each of thethree horizontal lines over the mean titers, the value indicated with avertical bar was compared pair-wise to each of the others and recordedas being significantly (*, p<0.05) or not significantly (ns) different.The detection limit for the neutralization assay is indicated with adotted line. ND, neutralization titer was below the detection limit.

FIGS. 54A and 54B. Protection against RSV challenge of hamstersimmunized with the indicated rB/HPIV3 vectors. The hamsters (n=6 animalsper immunization group) that had been immunized as shown in FIG. 53 werechallenged IN on day 30 post-immunization with 10⁶ PFU of wt RSV in a0.1 ml inoculum. On day 3 post-challenge, hamsters were euthanized and(A) nasal turbinates and (B) lungs were collected. RSV titers in tissuehomogenates were determined by plaque assay in Vero cells at 37° C. Eachsymbol represents an individual animal and mean values of viral titersof the groups are shown above the symbols and indicated as shorthorizontal lines. The pairwise student t-test was used to evaluate thestatistical significance of differences between values: in each of thehorizontal lines over the mean titers, the value(s) indicated with avertical bar(s) was compared pair-wise to each of the others andrecorded as being significantly (*, p<0.05) or not significantly (ns)different. The detection limit of the assay was log₁₀ 1.7 PFU/g oftissue, indicated as a dotted line.

FIG. 55. rB/HPIV3 constructs that were evaluated for attenuation andimmunogenicity in non-human primates (Rhesus macaques). Rhesus macaqueswere infected by the combined IN and intratracheal routes with 10⁶TCID₅₀ per site of the following constructs: HEK/GA-opt/DS/B3TMCT;HEK/GA-opt/DS-Cav1/B3TMCT; and HEK/GS-opt/DS-Cav1/B3TMCT in groups offour, six and six animals, respectively.

FIGS. 56A and 56B. Replication of rB/HPIV3 vectors in rhesus macaques.Rhesus macaques were infected with the rB/HPIV3 vectors indicated inFIG. 55. Vector replication in the respiratory tract was assessed bycollecting (A) nasopharyngeal swabs and (B) tracheal lavages on theindicated days and determining the viral titers by limiting dilutionassay. Limit of detection is 1.2 log₁₀ TCID₅₀/mL shown as dotted line.

FIGS. 57A and 57B. Serum RSV-neutralizing antibody titers induced byrB/HPIV3 vectors. From the experiment shown in FIGS. 55 and 56, serawere collected at 0, 14, 21, and 28 days post-immunization. FIG. 57A:RSV neutralizing antibody titers at the indicated time points weredetermined by a 60% plaque reduction neutralization test (PRNT₆₀) in thepresence of added guinea pig complement. The statistical significance ofdifference in mean titers of each time point was determined by pairwisestudent-t test (ns, P>0.05). FIG. 57A: RSV neutralizing antibody titersat day 28 post-immunization were determined by PRNT₆₀ in the absence ofadded complement. The detection limit for the neutralization assay isindicated with a dotted line. The statistical significance of differencein mean titers of each time point was determined by pairwise student-ttest (ns, P>0.05).

FIGS. 58A and 58B. Construction of a rB/HPIV3 vector expressingHEK/GS-opt/DS-Cav1/B3TMCT from the pre-N position, and modification ofthe amino acid sequence of the HPIV3 HN protein to achieve increasedphenotypic stability of the vector. FIG. 58A: Insertion of theHEK/GS-opt/DS-Cav1/B3TMCT insert into the first gene position ofrB/HPIV3. FIG. 58A: Corrections of the HPIV3 HN gene that conferredincreased phenotypic stability. The HN gene in the original recombinantHPIV3 made by reverse genetics (Durbin et al Virology 235:323-332 1997)had two engineered nucleotide substitutions in the HN gene at antigenomepositions 7913 and 7915 that resulted in the amino acid substitutionP370T, and an adventitious mutation at antigenome position 7593 thatresulted in the amino acid substitution T263I. Here, these mutationswere changed back to the “wild-type” assignments, i.e., that found inbiologically derived HPIV3 strain JS (Genbank Z11575.1; Stokes et alVirus Res 25:91-103. 1992).

FIGS. 59A and 59B. Intracellular expression of RSV F and vector proteinsby vectors expressing various versions of RSV F protein in the firstgene position (pre-N) or in the second gene position (N-P). Analysis ofrB/HPIV3-wt HN-HEK/GS-opt/DS-Cav1/B3TMCT/pre-N, the construct diagrammedin FIG. 58A. Vero (FIG. 59A) and LLC-MK2 (FIG. 59B) cells were infectedwith empty rB/HPIV3 vector (empty B/H3, lane 1), or thewtHN/HEK/GS-opt/DS-Cav1/B3TMCT/pre-N construct (pools CL20a, CL24a,lanes 3 and 4), or vector with the same version of RSV F inserted in thesecond (N-P) position (HEK/GS-opt/DS-Cav1/B3TMCT/N-P, lane 5), or vectorwith Non-HEK/non-opt version of RSV F inserted in the pre-N position(lane 6), or wt RSV (lane 2), or mock-infected (lane 7). The vectorswere infected at an MOI of 10 TCID₅₀/cell, and wt RSV at MOI of 3PFU/cell. Infected monolayers were incubated at 32° C. Cell lysates werecollected at 48 hpi and subjected to Western blot analysis. RSV F in theforms of cleaved F₁ and/or uncleaved F₀ were detected. BPIV3 N and Pproteins were used to evaluate effects on vector protein expression.GAPDH was used as a loading control.

FIG. 60. HPIV1 vector: sequences of the cytoplasmic tails (CT),transmembrane (TM) domains, and adjoining regions of the ectodomains ofthe RSV F protein (strain A2, amino acid assignments) and HPIV1 Fprotein (boldface), with the amino acid sequence positions indicated.RSV-F-TMCT is a chimeric protein consisting of the ectodomain of RSV Fprotein attached to the TM and CT domains of HPIV1 F protein. Thesequences shown are as follows: RSV(A2)F (SEQ ID NO: 1, residues510-574), HPIV1 F (SEQ ID NO: 152), RSV F-TMCT (SEQ ID NO: 135, residues510-588).

FIGS. 61 and 62. Construction of HPIV1-C^(Δ170) vectors expressingversions of RSV F protein designed to be stabilized in the prefusionconformation (DS-Cav1) and to have increased incorporation into theHPIV1 vector particle. Each of these modified RSV F inserts containedthe HEK assignments (HEK) and was codon-optimized by GS for humanexpression (GS-opt). The RSV F insert was engineered to be stabilized inthe prefusion conformation by the DS and Cav1 mutations (DS-Cav1) alone(upper construct in FIGS. 61 and 62) or with further modification by thereplacement of its TMCT domain with those from HPIV1 F (TMCT, lowerconstruct in FIGS. 61 and 62). The resulting HEK/GS-opt/DS-Cav1 andHEK/GS-opt/DS-Cav1/TMCT versions of RSV F were modified by flankingsequence and inserted into the HPIV1-C^(Δ170) vector (see Example 2 foran explanation of the HPIV1 vector and C^(Δ170) mutation) at the (FIG.61) first gene position (MluI site), or (FIG. 62) second gene position(AscI site). In each case, the RSV F was under the control of HPIV1transcription signals for expression as a separate mRNA. Nucleotidenumbering is relative to the complete antigenome RNA sequence of thefinal construct. The sequences of SEQ ID NOs: 146 and 147 are shownflanking the RSV F insert under the diagrams for F1/HEK/GS-opt/DS-Cav1,F1/HEK/GS-opt/DS-Cav1/TMCT, F2/HEK/GS-opt/DS-Cav1, andF2/HEK/GS-opt/DS-Cav1/TMCT.

FIG. 63. Kinetics of multi-cycle growth in Vero cells of rHPIV1-C^(Δ170)vectors expressing RSV F stabilized in the prefusion conformation(DS-Cav1), without or with TMCT from HPIV1 F protein. Vero cells wereinfected with the constructs in triplicate at an MOI of 0.01 andincubated for 7 days at 32 C. At 24 h intervals, 0.5 mL of the total 3mL culture supernatant was collected over 7 days. After samplecollection, 0.5 mL fresh media was added to each culture to restore theoriginal volume. Virus titration of the collected samples was performedon LLC-MK2 cells by hemadsorption assay and values are plotted asmeans±SEM.

FIG. 64. Incorporation into HPIV1-C^(Δ170) virion particles of RSV Fprotein stabilized in the prefusion conformation (DS-Cav1) without orwith TMCT from HPIV1 F protein. The indicated virus constructs (thedesignations HEK/GS-opt were omitted for the sake of brevity) were grownin LLC-MK2 cells and virions were purified by sucrose gradientcentrifugation. Protein concentration of the purified viruses wasdetermined by BCA assay. 1 μg total protein from each purified virus waslysed in RIPA lysis buffer, reduced, denatured, and subjected toSDS-PAGE and Western blot analysis. RSV F (top panel) and HPIV1 proteins(second, third, and fourth panels) were detected with mouse monoclonaland rabbit polyclonal HPIV1-peptide-specific (N, F, and HN) antibodies,respectively. Infared-labeled secondary antibodies were used to detectbound primary antibodies. The chimeric RSV-F-DS-Cav1/TMCT protein inlanes 2 and 4 (fourth panel) are visible because the antipeptide serumspecific to HPIV1 F protein was raised using a synthetic peptidecontaining the C-terminal 18 amino acids of the CT domain, and thusreacts with RSV F protein bearing the HPIV1 F protein TMCT domains.

FIG. 65. Expression in infected Vero cells of RSV F protein stabilizedin the prefusion conformation (DS-Cav1) without and with TMCT from HPIV1F protein. Vero cell monolayers in 6-well plates were inoculated withthe indicated viruses (the designations HEK/GS-opt were omitted for thesake of brevity) including the wt HPIV1 and rHPIV1-C^(Δ170) empty vectorcontrols at an MOI of 5 and incubated for 48 h at 32° C. Cell lysateswere prepared by lysing the monolayers in 200 μL LDS sample buffer.Protein samples were reduced and denatured, and 45 μL of each samplewere electrophoresed followed by protein transfer to PVDF membranes. RSVF and HPIV1 proteins were detected using the same primary and secondaryantibodies as described for FIG. 64.

FIG. 66. Sequences of the cytoplasmic tails (CT), transmembrane (TM)domains, and adjoining regions of the ectodomains of the RSV F protein(amino acid assignments) and HPIV3 F protein (boldface), with the aminoacid sequence positions indicated. RSV-F-H3TMCT is a chimeric proteinconsisting of the ectodomain of RSV F protein attached to the TM and CTdomains of HPIV3 F protein. The sequences shown are as follows: RSV(A2)F(SEQ ID NO: 1, residues 510-574), HPIV3 F (SEQ ID NO: 153), RSV F-H3TMCT(SEQ ID NO: 10, residues 510-575).

FIGS. 67A and 67B. Construction of rHPIV3 vectors expressing versions ofRSV F protein designed to be stabilized in the prefusion conformation(DS-Cav1) and to have increased incorporation into the rHPIV3 vectorparticle. The vector is wild type rHPIV3 strain JS which was modified tocontain the 263T and 370P amino acid assignments in the HN protein (seeFIG. 58B), which were found to confer phenotypic stability to thevector. In addition, the rHPIV3 vector was modified by the creation of aBlpI site at positions 103-109 (A, top construct), for insertion of RSVF (or potentially any other insert) in gene position 1, or the creationof an AscI site at positions 1675-1682 (B, top construct), for insertionof RSV F in gene position 2. Each of the modified RSV F insertscontained the HEK assignments (HEK) and was codon-optimized by GS forhuman expression (GS-opt). In addition, the RSV F insert was engineeredto be stabilized in the prefusion conformation by the DS and Cav1mutations (DS Cav1) alone (A and B, second construct) or with furthermodification by the replacement of its TMCT domains with those fromrHPIV3 F (H3TMCT, A and B, third construct). The resultingHEK/GS-opt/DS-Cav1 and HEK/GS-opt/DS-Cav1/H3TMCT versions of RSV F weremodified by flanking sequence and inserted into the (A) first geneposition (BlpI site), or (B) second gene position (AscI site) of wtrHPIV3 JS. In each case, the RSV F was under the control of HPIV3transcription signals for expression as a separate mRNA. Nucleotidenumbering is relative to the complete antigenome RNA sequence of thefinal construct. The sequence of SEQ ID NOs: 148 is shown under thediagram for rHPIV3 wt-JS. The sequences of SEQ ID NOs: 149 and 150 areshown flanking the RSV F insert under the diagrams forF1/HEK/GS-opt/DS-Cav1, F1/HEK/GS-opt/DS-Cav1/H3TMCT,F2/HEK/GS-opt/DS-Cav1, and F2/HEK/GS-opt/DS-Cav1/H3TMCT.

SEQUENCING LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜348 kb), which wascreated on Aug. 30, 2019 which is incorporated by reference herein. Inthe accompanying sequence listing:

DETAILED DESCRIPTION

A previous study (Zimmer et al J Virol 2005 79:10467-77) evaluated theexpression of RSV F protein from a heterologous gene in the Sendaivirus, which is a murine relative of HPIV1 and also is closely relatedto HPIV3. That study showed that very little RSV F protein wasincorporated into the Sendai virus vector particle. The investigatorsreplaced the CT or CT plus TM of the RSV F protein with thecorresponding sequences from the Sendai F protein on the premise thatthis would improve the efficiency of interaction of the foreign RSV Fprotein with the vector particle. These modifications indeed increasedincorporation of the engineered RSV F into the Sendai particle, but onlyif the Sendai F protein gene was also deleted. The requirement to deletethe vector F protein is incompatible with the generation of infectious,attenuated viruses for vaccination and also would remove one of thevector protective antigens, which are believed to be needed to generatea bivalent vaccine.

As disclosed herein, when expressed by rB/HPIV3, HPIV3, or HPIV1, theRSV F protein including RSV F TM and CT is incorporated into the vectorparticle only in trace amounts. However, swapping the TM and CT of theheterologous RSV F protein for the corresponding TM and CT of theparamyxovirus F protein provided a multi-fold increase in RSV Fectodomain incorporation in the envelope of recombinant paramyxovirus,such that the packaging of RSV F into the vector was as efficient (e.g.B/HPIV3) or more efficient (e.g. HPIV1) per μg of purified virion thanthat of RSV itself. This was effective when the TM and CT were swappedtogether, or when the CT was swapped alone. However, unexpected effectsof increased fusogenicity of the chimeric RSV F specific to CT aloneprovide guidance that TMCT is preferred.

Efficient packaging of RSV F into the vector particle dramaticallyincreased the elicitation of an immune response to the ectodomain(bearing all of the neutralization epitopes) when the recombinantparamyxovirus was administered to a subject. Unexpectedly, thevirus-neutralizing serum antibody response was dramatically increased inquality, which was assessed by comparing RSV-neutralization activity invitro in the absence of complement (which measures strongly-neutralizingantibodies) or in its presence (which augments neutralization by weak ornon-neutralizing antibodies). This unanticipated increase in antibodyquality is of particular importance for RSV, which is noted for inducingincomplete immune protection. The expression and efficient packaging ofa foreign glycoprotein bearing the TMCT domains of a vector glycoproteinhad the obvious potential to disrupt vector replication andmorphogenesis: however, constructs are provided in which this effect wasminimal.

To further increase immunogenicity, stabilization of the RSV F proteinin the pre-fusion conformation was evaluated. On its own, pre-fusionstabilization also resulted in an increase in titers ofstrongly-neutralizing antibodies, suggestive of stabilization ofneutralization epitopes. In the hamster model, the effect of pre-fusionstabilization on increased immunogenicity and protection appeared to beadditive to that of efficient packaging conferred by TMCT. However, whenevaluated in non-human primates, the effect of packaging appeared to begreater than that of pre-fusion stabilization.

Given the challenge of achieving protection against RSV, maximalimmunogenicity is desired. Extensive experimentation uncovered otheraspects of vector and insert construction (e.g., use of variousinsertion sites, use of codon-optimization, and use of an early-passageRSV F protein sequence) that provided increased expression of RSV F andreduced the cytopathic effects of syncytia formation mediated by thehighly fusogenic RSV F protein.

It is noteworthy that a prototype vaccine virus based on rB/HPIV3expressing an unmodified RSV F protein, which in clinical trials haddisappointing RSV immunogenicity (Bernstein, et al. 2012. PediatricInfectious Disease Journal 31:109-114), was confirmed by the methods ofthe present disclosure to induce RSV-neutralizing serum antibodies thatwere of poor quality, possessing neutralization activity in vitro onlyin the presence of added complement. In contrast, disclosed constructsinduced, in African green monkeys, high titers of serum antibodiescapable of efficiently neutralizing RSV in vitro in the absence ofcomplement.

I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion, for example, in whichantigen solution is emulsified in mineral oil (Freund incompleteadjuvant), sometimes with the inclusion of killed mycobacteria (Freund'scomplete adjuvant) to further enhance antigenicity (inhibits degradationof antigen and/or causes influx of macrophages). Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants. Adjuvants include biological molecules (a “biologicaladjuvant”), such as costimulatory molecules. Exemplary adjuvants includeIL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix, and toll-likereceptor (TLR) agonists, such as TLR-9 agonists, Poly I:C, or PolyICLC.The person of ordinary skill in the art is familiar with adjuvants (see,e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems.Wiley-Interscience, 2007). Adjuvants can be used in combination with thedisclosed recombinant.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intranasal, the composition (such as a compositionincluding a disclosed recombinant paramyxovirus) is administered byintroducing the composition into the nasal passages of the subject.Exemplary routes of administration include, but are not limited to,oral, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), sublingual, rectal, transdermal (forexample, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in apolypeptide with a different amino acid or with no amino acid (i.e., adeletion). In some examples, an amino acid in a polypeptide issubstituted with an amino acid from a homologous polypeptide, forexample, and amino acid in a recombinant group A RSV F polypeptide canbe substituted with the corresponding amino acid from a group B RSV Fpolypeptide. Reference to a “66E” amino acid in a RSV F protein refersto an RSV F protein comprising a glutamate residue at position 66. Theamino acid can be present due to substitution from a reference sequence.Reference to a “K66E” substitution in an RSV F protein refers to an RSVF protein comprising a glutamate residue at position 66 that has beensubstituted for a lysine residue in a reference (e.g., native) sequence.

Attenuated: A paramyxovirus that is “attenuated” or has an “attenuatedphenotype” refers to a paramyxovirus that has decreased virulencecompared to a reference wild type paramyxovirus under similar conditionsof infection. Attenuation usually is associated with decreased virusreplication as compared to replication of a reference wild-typeparamyxovirus under similar conditions of infection, and thus“attenuation” and “restricted replication” often are used synonymously.In some hosts (typically non-natural hosts, including experimentalanimals), disease is not evident during infection with a referenceparamyxovirus in question, and restriction of virus replication can beused as a surrogate marker for attenuation. In some embodiments, arecombinant paramyxovirus (e.g., RSV, PIV3) that is attenuated exhibitsat least about 10-fold or greater decrease, such as at least about100-fold or greater decrease in virus titer in the upper or lowerrespiratory tract of a mammal compared to non-attenuated, wild typevirus titer in the upper or lower respiratory tract, respectively, of amammal of the same species under the same conditions of infection.Examples of mammals include, but are not limited to, humans, mice,rabbits, rats, hamsters, such as for example Mesocricetus auratus, andnon-human primates, such as for example Ceroptihecus aethiops. Anattenuated paramyxovirus may display different phenotypes includingwithout limitation altered growth, temperature sensitive growth, hostrange restricted growth, or plaque size alteration.

Cytoplasmic Tail (CT): A contiguous region of a transmembrane proteinthat includes a terminus (either N- or C-terminus) of the protein andextends into the cytoplasm of a cell or enveloped virus from thecytoplasmic surface of the cell membrane or viral envelope. In the caseof a type I transmembrane protein, the CT includes the C-terminus of theprotein. In the case of a type II transmembrane protein, the CT includesthe N-terminus of the protein.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a polypeptidethat includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequencesencoding a peptide are included as long as the amino acid sequence ofthe peptide encoded by the nucleotide sequence is unchanged.

Gene: A nucleic acid sequence, typically a DNA sequence, that comprisescontrol and coding sequences necessary for the transcription of an RNA,whether an mRNA or otherwise. For instance, a gene may comprise apromoter, one or more enhancers or silencers, a nucleic acid sequencethat encodes a RNA and/or a polypeptide, downstream regulatory sequencesand, possibly, other nucleic acid sequences involved in regulation ofthe expression of an mRNA.

Heterologous: Originating from a different genetic source. Aheterologous gene included in a recombinant viral genome is a gene thatdoes not originate from that viral genome. In one specific, non-limitingexample, a heterologous gene encoding an ectodomain of a RSV F proteinis included in the genome of a recombinant PIV vector. Methods forintroducing a heterologous gene in a viral vector are well known in theart and also described herein.

Host cells: Cells in which a vector can be propagated and its nucleicacid expressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunogen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An immunogenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as a disclosedrecombinant paramyxovirus. Administration of an immunogen to a subjectcan lead to protective immunity against a pathogen of interest.

Immunogenic composition: A composition comprising an immunogen thatinduces a measurable T cell response against an antigen, or induces ameasurable B cell response (such as production of antibodies) against anantigen, included on the immunogen or encoded by a nucleic acid moleculeincluded in the immunogen. In one example, an immunogenic composition isa composition that includes a disclosed recombinant paramyxovirus thatinduces a measurable CTL response against RSV and/or PIV, or induces ameasurable B cell response (such as production of antibodies) againstRSV and/or PIV, when administered to a subject. An immunogeniccomposition can include an isolated recombinant paramyxovirus asdisclosed herein. For in vivo use, the immunogenic composition willtypically include a recombinant paramyxovirus in a pharmaceuticallyacceptable carrier and may also include other agents, such as anadjuvant.

Isolated: An “isolated” biological component has been substantiallyseparated or purified away from other biological components, such asother biological components in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA, RNA, and proteins.Proteins, peptides, nucleic acids, and viruses that have been “isolated”include those purified by standard purification methods. Isolated doesnot require absolute purity, and can include protein, peptide, nucleicacid, or virus molecules that are at least 50% isolated, such as atleast 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Linked: The terms “linked,” “linkage,” and “linking” refer to making twomolecules into one contiguous molecule; for example, linking twopolypeptides into one contiguous polypeptide by recombinant means.Reference to a gene encoding a type I membrane protein comprising a RSVF ectodomain “linked” to a TM and CT of a heterologous F protein refersto genetic linkage between the nucleic acid sequence encoding the RSV Fectodomain and the nucleic acid sequence encoding the TM and CT of theheterologous F protein in the gene by recombinant means, such thatexpression of the gene leads to production of a protein including, inthe N- to C-terminal direction, the RSV F ectodomain, the TM, and theCT. In some embodiments, the C-terminal residue of the RSV F ectodomaincan be directly linked (by peptide bond) to the N-terminal residue ofthe TM. In some embodiments, the C-terminal residue of the RSV Fectodomain can be indirectly linked to the N-terminal residue of the TMvia a peptide linker (such as a glycine-serine linker).

Linker: A bi-functional molecule that can be used to link two moleculesinto one contiguous molecule. Non-limiting examples of peptide linkersinclude glycine-serine linkers.

Native protein, sequence, or di-sulfide bond: A polypeptide, sequence ordi-sulfide bond that has not been modified, for example by selectivemutation. For example, selective mutation to focus the antigenicity ofthe antigen to a target epitope, or to introduce a di-sulfide bond intoa protein that does not occur in the native protein. Native protein ornative sequence are also referred to as wild-type protein or wild-typesequence. A non-native di-sulfide bond is a disulfide bond that is notpresent in a native protein, for example a di-sulfide bond that forms ina protein due to introduction of one or more cysteine residues into theprotein by genetic engineering.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked nucleic acid sequences arecontiguous and, where necessary to join two protein-coding regions, inthe same reading frame.

Paramyxovirus: A family of enveloped non-segmented negative-sensesingle-stranded RNA viruses. Examples of paramyxoviruses include, butare not limited to, human parainfluenza virus (HPIV) including types 1,2, 3, 4A, and 4B (HPIV1, HPIV2, HPIV3, HPIV4A, and HPIV4B,respectively), mouse parainfluenza type 1 (Sendai virus, MPIV1), bovineparainfluenza virus type 3 (BPIV3), parainfluenza virus 5 (PIV5,previously called simian virus 5, SV5), simian virus 41 (SV41), andmumps virus. HPIV1, HPIV3, MPIV1, and BPIV3 are classified in the genusRespirovirus. HPIV2, HPIV4, SV5, SV41, and mumps virus are classified inthe genus Rubulavirus. MPIV1, PIV5, and BPIV3 are animal relatives ofHPIV1, HPIV2, and HPIV3, respectively (Chancock et al., ParainfluenzaViruses, Knipe et al. (Eds.), pp. 1341-1379, Lippincott Williams &Wilkins, Philadelphia, 2001). HPIV1, HPIV2, and HPIV3 represent distinctserotypes and do not elicit significant cross immunity. HPIVs areetiological agents of respiratory infections such as croup, pneumonia,or bronchitis.

Parainfluenza virus (PIV): A number of enveloped non-segmentednegative-sense single-stranded RNA viruses from family Paramyxoviridaethat are descriptively grouped together. This includes all of themembers of genus respirovirus (e.g., HPIV1, HPIV3) and a number ofmembers of genus rubulavirus (e.g. HPIV2, HPIV4, PIV5). Members of genusavulavirus (e.g., NDV) historically have been called PIVs and can beconsidered as part of this group. HPIV serotypes 1, 2, and 3 are secondonly to RSV in causing severe respiratory infections in infants andchildren worldwide, with HPIV3 being the most important of the HPIVs interms of disease impact. PIVs are made up of two structural modules: (1)an internal ribonucleoprotein core, or nucleocapsid, containing theviral genome, and (2) an outer, roughly spherical lipoprotein envelope.The PIV viral genome is approximately 15,000 nucleotides in length andencodes at least eight polypeptides. These proteins include thenucleocapsid structural protein (NP, NC, or N depending on the genera),the phosphoprotein (P), the matrix protein (M), the fusion glycoprotein(F), the hemagglutinin-neuraminidase glycoprotein (HN), the largepolymerase protein (L), and the C and D proteins. The P gene containsone or more additional open reading frames (ORFs) encoding accessoryproteins. The gene order is 3′-N-P-M-F-HN-L-5′, and each gene encodes aseparate protein encoding mRNA. Exemplary PIV strain sequences are knownto the person of ordinary skill in the art, such as the sequences of theHPIV1, HPIV2, HPIV3, and BPIV3 viruses.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate. In particular embodiments, suitable foradministration to a subject the carrier may be sterile, and/or suspendedor otherwise contained in a unit dosage form containing one or moremeasured doses of the composition suitable to induce the desired immuneresponse. It may also be accompanied by medications for its use fortreatment purposes. The unit dosage form may be, for example, in asealed vial that contains sterile contents or a syringe for injectioninto a subject, or lyophilized for subsequent solubilization andadministration or in a solid or controlled release dosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues.

Prime-boost vaccination: An immunotherapy including administration of afirst immunogenic composition (the primer vaccine) followed byadministration of a second immunogenic composition (the booster vaccine)to a subject to induce an immune response. The booster vaccine isadministered to the subject after the primer vaccine; the skilledartisan will understand a suitable time interval between administrationof the primer vaccine and the booster vaccine, and examples of suchtimeframes are disclosed herein. Additional administrations can beincluded in the prime-boost protocol, for example a second boost.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring: for example, includes one ormore nucleic acid substitutions, deletions or insertions, and/or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination can beaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of isolated segments of nucleic acids, for example, bygenetic engineering techniques.

A recombinant virus is one that includes a genome that includes arecombinant nucleic acid molecule.

A recombinant protein is one that has a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. In several embodiments, arecombinant protein is encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell, or into the genome of arecombinant virus.

Respiratory Syncytial Virus (RSV): An enveloped non-segmentednegative-sense single-stranded RNA virus of the family Paramyxoviridae.The RSV genome is ˜15,000 nucleotides in length and includes 10 genesencoding 11 proteins, including the glycoproteins SH, G and F. The Fprotein mediates fusion, allowing entry of the virus into the cellcytoplasm and also promoting the formation of syncytia. Two antigenicsubgroups of human RSV strains have been described, the A and Bsubgroups, based primarily on differences in the antigenicity of the Gglycoprotein. RSV strains for other species are also known, includingbovine RSV. Exemplary RSV strain sequences are known to the person ofordinary skill in the art. Further, several models of human RSVinfection are available, including model organisms infected with hRSV,as well as model organisms infected with species specific RSV, such asuse of bRSV infection in cattle (see, e.g., Bern et al., Am J, Physiol.Lung Cell Mol. Physiol., 301: L148-L156, 2011; and Nam and Kun (Eds.).Respiratory Syncytial Virus: Prevention, Diagnosis and Treatment. NovaBiomedical Nova Science Publisher, 2011; and Cane (Ed.) RespiratorySyncytial Virus. Elsevier Science, 2007.)

RSV Fusion (F) protein: An RSV envelope glycoprotein that facilitatesfusion of viral and cellular membranes. In nature, the RSV F protein isinitially synthesized as a single polypeptide precursor approximately574 amino acids in length, designated F₀. F₀ includes an N-terminalsignal peptide that directs localization to the endoplasmic reticulum,where the signal peptide (approximately the first 22 residues of F₀) isproteolytically cleaved. The remaining F₀ residues oligomerize to form atrimer which is again proteolytically processed by a cellular proteaseat two conserved furin consensus cleavage sequences (approximately F₀positions 109/110 and 136/137; for example, RARR₁₀₉ (SEQ ID NO: 1,residues 106-109) and RKRR₁₃₆ (SEQ ID NO: 1, residues 133-136) to excisethe pep27 polypeptide and generate two disulfide-linked fragments, F₁and F2. The smaller of these fragments, F₂, originates from theN-terminal portion of the F₀ precursor and includes approximatelyresidues 26-109 of F₀. The larger of these fragments, F₁, includes theC-terminal portion of the F₀ precursor (approximately residues 137-574)including an extracellular/lumenal region (˜residues 137-529), a TM(residues 530-550), and a CT (residues 551-574) at the C-terminus.

Three F₂-F₁ protomers oligomerize in the mature F protein, which adoptsa metastable “prefusion” conformation that is triggered to undergo aconformational change (to a “postfusion” conformation) upon contact witha target cell membrane. This conformational change exposes a hydrophobicsequence, known as the fusion peptide, which is located at theN-terminus of the F₁ polypeptide, and which associates with the hostcell membrane and promotes fusion of the membrane of the virus, or aninfected cell, with the target cell membrane.

The extracellular portion of the RSV F protein is the RSV F ectodomain,which includes the F2 protein and the F₁ ectodomain. An RSV F ectodomaintrimer includes a protein complex of three RSV F ectodomains.

The RSV F protein adopts a “prefusion” conformation prior to triggeringof the fusogenic event that leads to transition of RSV F to thepostfusion conformation and following processing into a mature RSV Fprotein in the secretory system. The three-dimensional structure of anexemplary RSV F protein in a prefusion conformation is known, anddisclosed for example in WO2014160463, which is incorporated byreference herein. In the prefusion state, the RSV F protein includes anantigenic site at its membrane distal apex termed “antigenic site Ø,”that includes RSV F residues 62-69 and 196-209, and also includes theepitopes of the D25 and AM22 monoclonal antibodies. Thus, a recombinantRSV F protein stabilized in a prefusion conformation can be specificallybound by an antibody that binds the pre- but not post-fusionconformation of the RSV F protein, such as an antibody that specificallybinds to an epitope within antigenic site Ø, for example, the D25 orAM22 antibody. Additional RSV F prefusion specific antibodies includethe 5C4 and MPE8 antibodies.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs, orthologs, or variants of a polypeptide will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a peptide sequence that has 1166matches when aligned with a test sequence having 1554 amino acids is75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a polypeptide (such as a RSV F ectodomain) aretypically characterized by possession of at least about 75%, for exampleat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity counted over the full length alignment with theamino acid sequence of interest. Proteins with even greater similarityto the reference sequences will show increasing percentage identitieswhen assessed by this method, such as at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs and variants will typically possess at least80% sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85% or at least 90% or 95%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are available atthe NCBI website on the internet. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is entirely possible that strongly significant homologs couldbe obtained that fall outside of the ranges provided.

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, New York, 2012) and Ausubel etal. (In Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, through supplement 104, 2013). One example of a useful algorithmis PILEUP. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153, 1989. Using PILEUP, a reference sequence is compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps. PILEUP can be obtained fromthe GCG sequence analysis software package, e.g., version 7.0 (Devereauxet al., Nuc. Acids Res. 12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

As used herein, reference to “at least 90% identity” refers to “at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or even100% identity” to a specified reference sequence.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In a particular example, the subject is a newborn infant. In anadditional example, a subject is selected that is in need of inhibitingof an RSV infection. For example, the subject is either uninfected andat risk of RSV infection or is infected in need of treatment.

Transmembrane domain (TM): An amino acid sequence that spans a lipidbilayer, such as the lipid bilayer of a cell or virus or virus-likeparticle. A transmembrane domain can be used to anchor an antigen to amembrane. In some examples a transmembrane domain is a RSV Ftransmembrane domain.

Vaccine: A preparation of immunogenic material capable of stimulating animmune response, administered for the prevention, amelioration, ortreatment of infectious or other types of disease. The immunogenicmaterial may include attenuated or killed microorganisms (such asbacteria or viruses), or antigenic proteins, peptides or DNA derivedfrom them. An attenuated vaccine is a virulent organism that has beenmodified to produce a less virulent form, but nevertheless retains theability to elicit antibodies and cell-mediated immunity against thevirulent form. An inactivated (killed) vaccine is a previously virulentorganism that has been inactivated with chemicals, heat, or othertreatment, but elicits antibodies against the organism. Vaccines mayelicit both prophylactic (preventative or protective) and therapeuticresponses. Methods of administration vary according to the vaccine, butmay include inoculation, ingestion, inhalation or other forms ofadministration. Vaccines may be administered with an adjuvant to boostthe immune response.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s)that is operationally linked to the coding sequence of an antigen(s) ofinterest and can express the coding sequence. Non-limiting examplesinclude a naked or packaged (lipid and/or protein) DNA, a naked orpackaged RNA, a subcomponent of a virus or bacterium or othermicroorganism that may be replication-incompetent, or a virus orbacterium or other microorganism that may be replication-competent. Avector is sometimes referred to as a construct. Recombinant DNA vectorsare vectors having recombinant DNA. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. Viral vectors arerecombinant nucleic acid vectors having at least some nucleic acidsequences derived from one or more viruses.

II. Recombinant Viral Vectors

Recombinant paramyxoviruses are provided that include antigens frommultiple viral pathogens, and can be used to induce an immune responseto those viral pathogens. The recombinant paramyxoviruses include agenome encoding a heterologous gene. The recombinant paramyxovirusescomprise a genome comprising a heterologous gene encoding the ectodomainof a transmembrane protein (e.g., a viral glycoprotein) of aheterologous viral pathogen. The ectodomain can be linked to a CT, or aTM and a CT of an envelope protein from the paramyxovirus to allow forexpression of the ectodomain of the transmembrane protein from theheterologous virus on the paramyxovirus envelope. For example, therecombinant paramyxovirus can be a recombinant PIV comprising a genomecomprising a heterologous gene encoding the ectodomain of an RSV Fprotein linked to the TM and CT of the F protein from the PIV.Additional description of the recombinant paramyxovirus andmodifications thereof is provided herein.

The paramyxovirus genome includes genes encoding N, P, M, F, HN, and Lproteins. The genome also includes a genomic promoter and anti-promoter,with the order of promoter-N, P, M, F, HN, L-antipromoter. Theheterologous gene included in the genome of the recombinantparamyxovirus can be located at any position between genes of theparamyxovirus genome, or between the promoter and the N gene, or the Lgene and the antipromoter. The heterologous gene can be flanked byappropriate gene start and gene-end sequences to facilitate expressionfrom the viral genome. In a preferred embodiment, the heterologous genecan be located between the promoter and the N gene, or between the Ngene and the P gene.

In an embodiment, the heterologous gene included in the genome of therecombinant paramyxovirus encodes the ectodomain of a type Itransmembrane protein (e.g., a type I viral glycoprotein) linked to aCT, or TM and CT, of the F protein of the paramyxovirus. In otherembodiments, the heterologous gene included in the genome of therecombinant paramyxovirus encodes the ectodomain of a type IItransmembrane protein (e.g., a type II viral glycoprotein) linked to aCT, or TM and CT, of the HN protein of the paramyxovirus.

The recombinant paramyxovirus can be a recombinant HPIV1, a HPIV2, aHPIV3, a BPIV3, a PIV5, a Sendai virus, or a NDV, or a chimera thereof,for example. Additional description of such recombinant paramyxovirus isprovided below.

General methods of generating a recombinant paramyxovirus including agenome including a heterologous gene are known to the person of ordinaryskill in the art, as are viral sequences and reagents for use in suchmethods. Non-limiting examples of methods of generating a recombinantPIV vector (such as a recombinant HPIV1, HPIV2, HPIV3, or H/BPIV3vector) including a heterologous gene, methods of attenuating thevectors (e.g., by recombinant or chemical means), as well as viralsequences and reagents for use in such methods are provided in US PatentPublications 2012/0045471; 2010/0119547; 2009/0263883; 2009/0017517;U.S. Pat. Nos. 8,084,037; 6,410,023; 8,367,074; 7,951,383; 7,820,182;7,704,509; 7,632,508; 7,622,123; 7,250,171; 7,208,161; 7,201,907;7,192,593, and Newman et al. 2002. Virus genes 24:77-92, Tang et al.,2003. J Virol, 77(20):10819-10828; each of which is incorporated byreference herein in its entirety. Non-limiting examples of methods ofgenerating a recombinant NDV vector including a heterologous gene, aswell as viral sequences and reagents for use in such methods areprovided in US Patent Publications 2012/0064112; and Basavarajappa etal. 2014 Vaccine, 32: 3555-3563, and McGinnes et al., J. Virol., 85:366-377, 2011, each of which is incorporated by reference herein in itsentirety. Non-limiting examples of methods of generating a recombinantSendai vector including a heterologous gene, as well as viral sequencesand reagents for use in such methods are provided in US PatentPublications 20140186397, and Jones et al., Vaccine, 30:959-968, 2012,each of which is incorporated by reference herein in its entirety.

A. HPIV1 Vectors

In some embodiments, the recombinant paramyxovirus can be a recombinantHPIV1 including a viral genome encoding HPIV1 N, P, C, M, F, HN, and Lproteins. Nucleic acid sequences of HPIV1 genomes, and the genestherein, are known in the art, as are structural and functional geneticelements that control gene expression, such as gene start and gene endsequences and viral genome and anti-genome promoters. An exemplary HPIV1Washington/1964 strain genome sequence is provided as GenBank Acc. No.AF457102.1, which is incorporated by reference herein in its entirety.This exemplary HPIV1 Washington/1964 strain genome sequence encodes N,P, C, M, F, HN, and L proteins set forth as:

-   HPIV1 N, SEQ ID NO: 24 (GenBank protein ID #AAL89400.1, incorporated    by reference herein)-   HPIV1 P, SEQ ID NO: 25 (GenBank protein ID #AAL89402.1, incorporated    by reference herein)-   HPIV1 C, SEQ ID NO: 26 (ORF of P, GenBank protein ID #AAL89403.1,    incorporated by reference herein)-   HPIV1 M, SEQ ID NO: 27 (GenBank protein ID #AAL89406.1, incorporated    by reference herein)-   HPIV1 F, SEQ ID NO: 28 (GenBank protein ID #AAL89407.1, incorporated    by reference herein)-   HPIV1 HN, SEQ ID NO: 29 (GenBank protein ID #AAL89408.1,    incorporated by reference herein)-   HPIV1 L, SEQ ID NO: 30 (GenBank protein ID #AAL89409.1, incorporated    by reference herein)

The corresponding gene-start and gene-end sequences for these HPIV1genes are provided below:

SEQ Gene Gene start SEQ ID Gene end ID Intergenic N agggttaaag 54aagtaagaaaaa 55 ctt P agggtgaatg 56 Aattaagaaaaa 57 ctt M agggtcaaag 58Aaataagaaaaa 59 ctt F agggacaaag 60 Aagtaagaaaaa 55 ctt HN agggttaaag 61Gaataagaaaaa 62 ctt L agggttaatg 63 Tagtaagaaaaa 64 ctt

Further, viral leader/genome promoter and trailer/antigenome promoter ofthe HPIV2 V94 strain as set forth in GenBank Acc. No. AF457102.1 asnucleotides 1-96 and 15544-15600, respectively.

The recombinant paramyxovirus can be a recombinant HPIV1 including aviral genome encoding HPIV1 N, P, C, M, F, HN, and L proteins as setforth above, or encoding HPIV1 N, P, C, M, F, HN, and L proteinsindividually having at least 90% (such as at least 95%) sequenceidentity to the HPIV1 N, P, C, M, F, HN, and L proteins set forth above.

In some embodiments the recombinant paramyxovirus can be a recombinantHPIV1 including a genome including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a HPIV1 F protein TM and CT as setforth below, or encoding a recombinant viral glycoprotein ectodomainfrom a type I membrane protein (such as RSV F ectodomain) linked to aHPIV1 F protein TM and CT having at least 90% (such as at least 95%)sequence identity to the HPIV1 F protein TM and CT as set forth below.In some embodiments the recombinant paramyxovirus can be a recombinantHPIV1 including a genome including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a HPIV1 F protein CT as set forthbelow, or encoding a recombinant viral glycoprotein ectodomain from atype I membrane protein (such as RSV F ectodomain) linked to a HPIV1 Fprotein CT having at least 90% (such as at least 95%) sequence identityto the HPIV1 F protein CT as set forth below. HPIV1 F protein TM and CTsequences are known (see, e.g., GenBank accession #AF457102.1,incorporated by reference herein). Exemplary HPIV1 F protein TM and CTsequences are set forth as:

HPIV1 F TM: QIIMIIIVCILIIIICGILYYLY, residues 1-23 of SEQ ID NO: 31HPIV1 F CT: RVRRLLVMINSTHNSPVNAYTLESRMRNPYMGNNSN, residues 24-59 of SEQID NO: 31 HPIV1 F TM + CT: QIIMIIIVCILIIIICGILYYLYRVRRLLVMINSTHNSPVNAYTLESRMRNPYMGNNSN, SEQ ID NO: 31B. HPIV2 Vectors

In some embodiments the recombinant paramyxovirus vector can be arecombinant HPIV2 including a viral genome encoding HPIV2 N, P, V, M, F,HN, and L proteins. The nucleic acid sequences of the gene encodingthese HPIV2 proteins are known in the art, as are structural andfunctional genetic elements that control gene expression, such as genestart and gene-end sequences and viral genome and anti-genome promoters.An exemplary HPIV2 V94 strain genome sequence is provided as GenBankAcc. No. AF533010.1, which is incorporated by reference herein in itsentirety. This exemplary HPIV2 V94 strain genome sequence encodes N, P,V, M, F, HN, and L proteins set forth as:

-   HPIV2 N, SEQ ID NO: 32 (encoded by GenBank No. AF533010.1,    incorporated by reference herein)-   HPIV2 P, SEQ ID NO: 33 (encoded by GenBank No. AF533010.1,    incorporated by reference herein)-   HPIV2 V, SEQ ID NO: 34 (ORF of P, encoded by GenBank No. AF533010.1,    incorporated by reference herein)-   HPIV2 M, SEQ ID NO: 35 (encoded by GenBank No. AF533010.1,    incorporated by reference herein)-   HPIV2 F, SEQ ID NO: 36 (encoded by GenBank No. AF533010.1,    incorporated by reference herein)-   HPIV2 HN, SEQ ID NO: 37 (encoded by GenBank No. AF533010.1,    incorporated by reference herein)-   HPIV2 L, SEQ ID NO: 38 (encoded by GenBank No. AF533010.1,    incorporated by reference herein)

The corresponding gene-start and gene end sequences for these HPIV2genes are provided below:

SEQ Gene Gene start SEQ ID Gene end SEQ ID Intergenic ID NAgattccggtgccg 65 aatttaagaaaaaa 66 acat P aggcccggacgggttag 67aatttaataaaaaa 68 ttaaaagaagttaagtaaaatttaaagaacacaat 117 agagaaaacct MAggtccgaaagc 69 aatctaacaaaaaaa 70 ctaaacattcaataataaatcaaagttc 118 FAggccaaattat 71 aatttaagaaaaaa 72 cctaaaat 119 HN Aagcacgaaccc 73tatttaagaaaaaa 74 taatctttatataatgtaacaatactactaagattata 120 atat LAggccaga 75 tatttaagaaaaa 76

Further, viral leader/genome promoter and trailer/antigenome promoter ofthe HPIV2 V94 strain as set forth in GenBank Acc. No. AF533010.1 are setforth as nucleotides 1-175 and 15565-15654, respectively.

The recombinant paramyxovirus can be a recombinant HPIV2 including aviral genome encoding HPIV2 N, P, V, M, F, HN, and L proteins as setforth above, or encoding HPIV2 N, P, V, M, F, HN, and L proteinsindividually having at least 90% (such as at least 95%) sequenceidentity to the HPIV2 N, P, V, M, F, HN, and L proteins set forth above.

In some embodiments the recombinant paramyxovirus can be a recombinantHPIV2 including a genome including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a HPIV2 F protein TM and CT as setforth below, or encoding a recombinant viral glycoprotein ectodomainfrom a type I membrane protein (such as RSV F ectodomain) linked to aHPIV2 F protein TM and CT having at least 90% (such as at least 95%)sequence identity to the HPIV2 F protein TM and CT as set forth below.In some embodiments the recombinant paramyxovirus can be a recombinantHPIV2 including a genome including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a HPIV2 F protein CT as set forthbelow, or encoding a recombinant viral glycoprotein ectodomain from atype I membrane protein (such as RSV F ectodomain) linked to a HPIV2 Fprotein CT having at least 90% (such as at least 95%) sequence identityto the HPIV2 F protein CT as set forth below. HPIV2 F protein TM and CTsequences are known (see, e.g., GenBank accession #AF533010.1,incorporated by reference herein). Exemplary HPIV2 F protein TM and CTsequences from the HPIV3 JS strain are set forth as:

HPIV2 F TM domain: TLYSLSAIALILSVITLVVVGLLIAYII, residues 1-28 of SEQ IDNO: 39 HPIV2 F CT: KLVSQIHQFRALAATTMFHRENPAVFSKNNHGNIYGIS, residues29-66 of SEQ ID NO: 39 HPIV2 F TM + CT:TLYSLSAIALILSVITLVVVGLLIAYIIKLVSQ IHQFRALAATTMFHRENPAVFSKNNHGNIYGIS, SEQID NO: 39C. HPIV3 Vectors

In some embodiments the recombinant paramyxovirus can be a recombinantHPIV3 including a viral genome encoding HPIV3 N, P, C, M, F, HN, and Lproteins. The nucleic acid sequences of the gene encoding these HPIV3proteins are known in the art, as are structural and functional geneticelements that control gene expression, such as gene start and gene endsequences and viral genome and anti-genome promoters. An exemplary HPIV3JS strain genome sequence is provided as GenBank Acc. No. Z11575, whichis incorporated by reference herein in its entirety. For this exemplaryHPIV3 JS strain genome sequence nucleic acid sequences encoding the N,P, C, M, F, HN, and L proteins are set forth below.

-   HPIV3 N, SEQ ID NO: 40 (encoded by nucleotides 111-1658 of GenBank    No. Z11575, incorporated by reference herein)-   HPIV3 P, SEQ ID NO: 41 (encoded by nucleotides 1784-3595 of GenBank    No. Z11575, incorporated by reference herein)-   HPIV3 C, SEQ ID NO: 114 (encoded by nucleotides 1794-2393 of GenBank    No. Z11575, incorporated by reference herein)-   HPIV3 M, SEQ ID NO: 42 (encoded by nucleotides 3753-4814 of GenBank    No. Z11575, incorporated by reference herein),-   HPIV3 F, SEQ ID NO: 43 (encoded by nucleotides 5072-6691 of GenBank    No. Z11575, incorporated by reference herein),-   HPIV3 HN, SEQ ID NO: 44 (encoded by nucleotides 6806-8524 of GenBank    No. Z11575, incorporated by reference herein)-   HPIV3 L, SEQ ID NO: 45 (encoded by nucleotides 8646-15347 of GenBank    No. Z11575, incorporated by reference herein)

In some embodiments, the HN gene in HPIV3 vector encodes a HPIV3 HNprotein comprising the amino acid sequence set forth as

(SEQ ID NO: 101) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFIIVLINSIKSEKAHESLLQDINNEFMEITEKIQMASDNTNDLIQSGVNTRLLTIQSHVQNYIPISLTQQMSDLRKFISEITIRNDNQEVLPQRITHDVGIKPLNPDDFWRCTSGLPSLMKTPKIRLMPGPGLLAMPTTVDGCVRTPSLVINDLIYAYTSNLITRGCQDIGKSYQVLQIGIITVNSDLVPDLNPRISHTFNINDNRKSCSLALLNIDVYQLCSTPKVDERSDYASSGIEDIVLDIVNYDGSISTTRFKNNNISFDQPYAALYPSVGPGIYYKGKIIFLGYGGLEHPINENVICNTTGCPGKTQRDCNQASHSTWFSDRRMVNSIIVVDKGLNSIPKLKVWTISMRQNYWGSEGRLLLLGNKIYIYTRSTSWHSKLQLGIIDITDYSDIRIKWTWHNVLSRPGNNECPWGHSCPDGCITGVYTDAYPLNPTGSIVSSVILDSQKSRVNPVITYSTATERVNELAILNRTLSAGYTTTSCITHYNKGYCFHIVEINHKSLNTFQPMLFKTEIPKSCS

An exemplary DNA sequence encoding SEQ ID NO: 101 is provided asfollows:

(SEQ ID NO: 102) atggaatactggaagcataccaatcacggaaaggatgctggtaatgagctggagacgtctatggctactcatggcaacaagctcactaataagataatatacatattatggacaataatcctggtgttattatcaatagtcttcatcatagtgctaattaattccatcaaaagtgaaaaggcccacgaatcattgctgcaagacataaataatgagtttatggaaattacagaaaagatccaaatggcatcggataataccaatgatctaatacagtcaggagtgaatacaaggcttcttacaattcagagtcatgtccagaattacataccaatatcattgacacaacagatgtcagatcttaggaaattcattagtgaaattacaattagaaatgataatcaagaagtgctgccacaaagaataacacatgatgtaggtataaaacctttaaatccagatgatttttggagatgcacgtctggtcttccatctttaatgaaaactccaaaaataaggttaatgccagggccgggattattagctatgccaacgactgttgatggctgtgttagaactccgtctttagttataaatgatctgatttatgcttatacctcaaatctaattactcgaggttgtcaggatataggaaaatcatatcaagtcttacagatagggataataactgtaaactcagacttggtacctgacttaaatcctaggatctctcatacctttaacataaatgacaataggaagtcatgttctctagcactcctaaatatagatgtatatcaactgtgttcaactcccaaagttgatgaaagatcagattatgcatcatcaggcatagaagatattgtacttgatattgtcaattatgatggttcaatctcaacaacaagatttaagaataataacataagctttgatcaaccatatgctgcactatacccatctgttggaccagggatatactacaaaggcaaaataatatttctcgggtatggaggtcttgaacatccaataaatgagaatgtaatctgcaacacaactgggtgccccgggaaaacacagagagactgtaatcaagcatctcatagtacttggttttcagataggaggatggtcaactccatcattgttgttgacaaaggcttaaactcaattccaaaattgaaagtatggacgatatctatgcgacaaaattactgggggtcagaaggaaggttacttctactaggtaacaagatctatatatatacaagatctacaagttggcatagcaagttacaattaggaataattgatattactgattacagtgatataaggataaaatggacatggcataatgtgctatcaagaccaggaaacaatgaatgtccatggggacattcatgtccagatggatgtataacaggagtatatactgatgcatatccactcaatcccacagggagcattgtgtcatctgtcatattagactcacaaaaatcgagagtgaacccagtcataacttactcaacagcaaccgaaagagtaaacgagctggccatcctaaacagaacactctcagctggatatacaacaacaagctgcattacacactataacaaaggatattgttttcatatagtagaaataaatcataaaagcttaaacacatttcaacccatgttgttcaaaacagagat tccaaaaagctgcagttaa

The corresponding gene-start and gene end sequences for these HPIV3genes are provided below:

SEQ Gene Gene start SEQ ID Gene end ID N aggattaaagac 77 aaataagaaaaa 78P Aggattaaag 79 aaataagaaaaa 80 M Aggattaaag 81 aaataaaggataatcaaaaa 82F Aggacaaaag 83 aattataaaaaa 84 HN Aggagtaaag 85 aaatataaaaaa 86 LAggagcaaag 87 aaagtaagaaaaa 88Further, viral genome and anti-genome promoters of the HPIV3 JS strainas set forth in GenBank Acc. No. Z11575 are provided as nucleotides 1-96(genomic promoter) and nucleotides 15367-15462 (antigenomic promoter),respectively.

The recombinant paramyxovirus can be a recombinant HPIV3 including aviral genome encoding HPIV3 N, P, C, M, F, HN, and L proteins as setforth above, or encoding HPIV3 N, P, C, M, F, HN, and L proteinsindividually having at least 90% (such as at least 95%) sequenceidentity to the HPIV3 N, P, C, M, F, HN, and L proteins set forth above.

In some embodiments the recombinant paramyxovirus can be a recombinantHPIV3 including a genome including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a HPIV3 F protein TM and CT as setforth below, or encoding a recombinant viral glycoprotein ectodomainfrom a type I membrane protein (such as RSV F ectodomain) linked to aHPIV3 F protein TM and CT having at least 90% (such as at least 95%)sequence identity to the HPIV3 F protein TM and CT as set forth below.In some embodiments the recombinant paramyxovirus can be a recombinantHPIV3 including a genome including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a HPIV3 F protein CT as set forthbelow, or encoding a recombinant viral glycoprotein ectodomain from atype I membrane protein (such as RSV F ectodomain) linked to a HPIV3 Fprotein CT having at least 90% (such as at least 95%) sequence identityto the HPIV3 F protein CT as set forth below. HPIV3 F protein TM and CTsequences are known (see, e.g., protein encoded by nucleotides 5072-6691of GenBank No. Z11575). Exemplary HPIV3 F protein TM and CT sequencesfrom the HPIV3 JS strain are set forth as:

HPIV3 F TM domain: IIIILIMIIILFIINITIITIAI, residues 1-23 of SEQ ID NO:46 HPIV3 F CT: KYYRIQKRNRVDQNDKPYVLTNK, residues 24-46 of SEQ ID NO: 46HPIV3 F TM + CT: IIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK, SEQ IDNO: 46D. Bovine PIV3 and Chimeric Human/Bovine PIV3 Vectors

In some embodiments the recombinant paramyxovirus can be bovine PIV3(BPIV3) or a chimeric paramyxovirus including a viral genome encoding acombination of N, P, C, V, M, F, HN, and L proteins from BPIV3 andHPIV3. For example, the chimeric viral genome can encode HPIV3 F and HNproteins and BPIV3 N, P, C, V, M, and L proteins. The nucleic acidsequences of the genes encoding these HPIV3 and BPIV3 proteins are knownin the art, as are structural and functional genetic elements thatcontrol gene expression, such as gene start and gene end sequences andviral genome and anti-genome promoters. An exemplary BPIV3 Kansas genomesequence is provided as GenBank Acc. No. AF178654, which is incorporatedby reference herein in its entirety. This exemplary BPIV3 Kansas straingenome sequence encodes N, P, C, V, M, F, HN, and L proteins set forthbelow:

-   BPIV3 N, SEQ ID NO: 47 (GenBank Acc. No.: AAF28254, encoded by    nucleotides 111-1658 of GenBank No. AF178654, each of which is    incorporated by reference herein)-   BPIV3 P, SEQ ID NO: 48 (GenBank Acc. No.: AAF28255, encoded by    nucleotides 1784-3574 of GenBank No. AF178654, each of which is    incorporated by reference herein)-   BPIV3 C, SEQ ID NO: 115 (encoded by nucleotide 1794-2399 of GenBank    No. AF178654, incorporated by reference herein)-   BPIV3 V, SEQ ID NO: 116 (encoded by nucleotide 1784-3018 of GenBank    No. AF178654 with an inserted nucleotide g between nucleotide    2505-2506 at a gene editing site located at nucleotide 2500-2507)-   BPIV3 M, SEQ ID NO: 49 (GenBank Acc. No.: AAF28256, encoded by    nucleotides 3735-4790 of GenBank No. AF178654, each of which is    incorporated by reference herein)-   BPIV3 F, SEQ ID NO: 50 (GenBank Acc. No.: AAF28257, encoded by    nucleotides 5066-6688 of GenBank No. AF178654, each of which is    incorporated by reference herein)-   BPIV3 HN, SEQ ID NO: 51 (GenBank Acc. No.: AAF28258, encoded by    nucleotides 6800-8518 of GenBank No. AF178654, each of which is    incorporated by reference herein)-   BPIV3 L, SEQ ID NO: 52 (GenBank Acc. No.: AAF28259, encoded by    nucleotides 8640-15341 of GenBank No. AF178654, each of which is    incorporated by reference herein)

In some embodiments, the HPIV3 HN gene included in chimeric B/HPIV3vector encodes a HPIV3 HN protein comprising the amino acid sequence setforth as SEQ ID NO: 101 or SEQ ID NO: 44, or a variant thereof. Anexemplary DNA sequence encoding SEQ ID NO: 101 is provided SEQ ID NO:102.

In some embodiments, the chimeric B/HPIV3 vector can include a HPIV3 Fgene in place of the

BPIV3 F gene, for example a gene encoding a HPIV3 F amino acid sequenceset forth as SEQ ID NO: 43, or a variant thereof.

The corresponding gene-start and gene end sequences for these BPIV3genes are provided below:

Gene Gene start SEQ ID Gene end SEQ ID N Aggattaaagaa 89 caagtaagaaaaa90 P Aggattaatgga 91 tgattaagaaaaa 92 M Aggatgaaagga 93 gaaaaatcaaaaa 94F Aggatcaaaggg 95 aaaagtacaaaaaa 96 HN Aggaacaaagtt 97 gaaataataaaaaa 98L Aggagaaaagtg 99 aaagtaagaaaaa 100Further, viral genome and anti-genome promoters of the BPIV3 Kansasstrain as set forth in GenBank Acc. No. AF178654 are provided asnucleotides 1-96 (genomic promoter) and nucleotides 15361-15456(antigenomic promoter), respectively.

The recombinant paramyxovirus including a viral genome encoding N, P, C,V, M, F, HN, and L proteins from HPIV3 and BPIV3 viruses can encode amixture of the HPIV3 and BPIV3 N, P, C, V, M, F, HN, and L proteins asset forth above, or can encode a mixture of the BPIV3 and HPIV3 N, P, C,V, M, F, HN, and L proteins individually having at least 90% (such as atleast 95%) sequence identity to the BPIV3 or HPIV3 N, P, C, V, M, F, HN,and L proteins set forth above.

In some embodiments, the recombinant paramyxovirus can include a viralgenome encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and Lproteins as set forth above, or encoding HPIV3 F and HN proteins andBPIV3 N, P, C, V, M, and L proteins individually having at least 90%(such as at least 95%) sequence identity to the corresponding HPIV3 Fand HN protein or BPIV3 N, P, C, V, M, and L protein set forth above.

In some embodiments, the recombinant paramyxovirus including a genomeencoding N, P, C, V, M, F, HN, and L proteins from BPIV3 can furtherinclude a heterologous gene encoding a recombinant viral glycoproteinectodomain from a type I membrane protein (such as RSV F ectodomain)linked to a TM and CT of the BPIV3 F protein as set forth below, orlinked to a TM and CT having at least 90% (such as at least 95%)sequence identity to the TM and CT of the BPIV3 F protein as set forthbelow. In some embodiments, the recombinant paramyxovirus including agenome encoding N, P, C, V, M, F, HN, and L proteins from BPIV3 canfurther include a heterologous gene encoding a recombinant viralglycoprotein ectodomain from a type I membrane protein (such as RSV Fectodomain) linked to a CT of the BPIV3 F protein as set forth below, orlinked to a CT having at least 90% (such as at least 95%) sequenceidentity to the CT of the BPIV3 F protein as set forth below.

In some embodiments, the recombinant paramyxovirus including a genomeencoding N, P, C, V, M, F, HN, and L proteins from HPIV3 and BPIV3viruses (such as HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and Lproteins) can further include a heterologous gene encoding a recombinantviral glycoprotein ectodomain from a type I membrane protein (such asRSV F ectodomain) linked to a TM and CT of the BPIV3 F protein as setforth below, or linked to a TM and CT having at least 90% (such as atleast 95%) sequence identity to the TM and CT of the BPIV3 F protein asset forth below. In some embodiments, the recombinant paramyxovirusincluding a genome encoding N, P, C, V, M, F, HN, and L proteins fromHPIV3 and BPIV3 viruses can further include a heterologous gene encodinga recombinant viral glycoprotein ectodomain from a type I membraneprotein (such as RSV F ectodomain) linked to a CT of the BPIV3 F proteinas set forth below, or linked to a CT having at least 90% (such as atleast 95%) sequence identity to the CT of the BPIV3 F protein as setforth below. Exemplary BPIV3 F protein TM and CT sequences from theBPIV3 Kansas strain are set forth as:

BPIV3 F TM domain: ITIIIVMIIILVIINITIIVV, residues 1-21 of SEQ ID NO: 53BPIV3 F CT: IIKFHRIQGKDQNDKNSEPYILTNRQ, residues 22-57 of SEQ ID NO: 53BPIV3 F TM + CT: ITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ, SEQ IDNO: 53

In some embodiments, the recombinant paramyxovirus including a genomeencoding N, P, C, V, M, F, HN, and L proteins from HPIV3 and BPIV3viruses (such as HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and Lproteins) can further include a heterologous gene encoding a recombinantviral glycoprotein ectodomain from a type I membrane protein (such asRSV F ectodomain) linked to a TM and CT of the HPIV3 F protein as setforth above, or linked to a TM and CT having at least 90% (such as atleast 95%) sequence identity to the TM and CT of the HPIV3 F protein asset forth above. In some embodiments, the recombinant paramyxovirusincluding a genome encoding N, P, C, V, M, F, HN, and L proteins fromHPIV3 and BPIV3 viruses can further include a heterologous gene encodinga recombinant viral glycoprotein ectodomain from a type I membraneprotein (such as RSV F ectodomain) linked to a CT of the HPIV3 F proteinas set forth below, or linked to a CT having at least 90% (such as atleast 95%) sequence identity to the CT of the HPIV3 F protein as setforth below.

E. Sendai Virus

In an embodiment, the recombinant paramyxovirus can be a recombinantSendai virus including a recombinant viral genome encoding Sendai virusN, P, C, V, M, F, HN, and L proteins including a heterologous geneencoding a recombinant viral glycoprotein ectodomain from a type Imembrane protein (such as RSV F ectodomain) linked to a TM and CT of theSendai virus F protein, or linked to a TM and CT having at least 90%(such as at least 95%) sequence identity to the CT of the Sendai virus Fprotein. In an embodiment, the recombinant paramyxovirus can be arecombinant Sendai virus including a recombinant viral genome encodingSendai virus N, P, C, V, M, F, HN, and L proteins including aheterologous gene encoding a recombinant viral glycoprotein ectodomainfrom a type I membrane protein (such as RSV F ectodomain) linked to a CTof the Sendai virus F protein, or linked to a CT having at least 90%(such as at least 95%) sequence identity to the CT of the Sendai virus Fprotein. Sendai virus F protein TM and CT sequences are known (see,e.g., GenBank accession #BAN84670, incorporated by reference herein).Exemplary Sendai virus F protein TM and CT sequences are set forth as:

Sendai F TM domain: VITIIVVMVVILVVIIVIIIV (residues 1-21 of SEQ ID NO:103) Sendai F CT: LYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR (residues22-65 of SEQ ID NO: 103) Sendai F TM + CT:VITIIVVMVVILVVIIVIIIVLYRLRRSMLMGNPDDRIPRDTYTLEPKI RHMYTNGGFDAMAEKR, SEQID NO: 103F. NDV

In some embodiments the recombinant paramyxovirus can be a recombinantNDV virus including a recombinant viral genome encoding NDV N, P, V, M,F, HN, and L proteins including a heterologous gene encoding arecombinant viral glycoprotein ectodomain from a type I membrane protein(such as RSV F ectodomain) linked to a TM and CT of the NDV F protein asset forth below, or linked to a TM and CT having at least 90% (such asat least 95%) sequence identity to the TM and CT of the NDV F protein asset forth below. In some embodiments the recombinant paramyxovirus canbe a recombinant NDV virus including a recombinant viral genome encodingNDV N, P, V, M, F, HN, and L proteins including a heterologous geneencoding a recombinant viral glycoprotein ectodomain from a type Imembrane protein (such as RSV F ectodomain) linked to a CT of the NDV Fprotein as set forth below, or linked to a CT having at least 90% (suchas at least 95%) sequence identity to the CT of the NDV F protein as setforth below. NDV virus F protein TM and CT sequences are known (see,e.g., GenBank accession #AAC28374, incorporated by reference herein).Exemplary NDV virus F protein TM and CT sequences are set forth as:

NDV F TM domain: IVLTIISLILVFGILSLILACYL (residues 1-21 of SEQ ID NO:104) NDV F CT: MYKQKAQQKTLLWLGNNTLDQMRATTKM (residues 22-49 of SEQ IDNO: 104) NDV F TM + CT:IVLTIISLVFGILSLILACYLMYKQKAQQKTLLWLGNNTLDQMRATTKM, SEQ ID NO: 104G. Heterologous Genes

The recombinant paramyxovirus vector includes a recombinant genomeincluding one or more heterologous genes encoding an ectodomain of oneor more heterologous envelope proteins (or antigenic fragment thereof)of a heterologous viral pathogen, wherein the ectodomain is linked to aTM and CT of an envelope protein from the recombinant paramyxovirus. Forexample, one or more heterologous envelope proteins (or antigenicfragment thereof) from measles virus, subgroup A or subgroup Brespiratory syncytial viruses, mumps virus, human papilloma viruses,type 1 or type 2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses, human metapneumovirus, ebolavirues (such as Zaire ebola virus), influenza viruses, or highlypathogenic coronaviruses (SARS, MERS) can be expressed by the disclosedrecombinant paramyxovirus. Examples of useful envelope proteins include,but are not limited to, measles virus HA and F proteins, subgroup A orsubgroup B respiratory syncytial virus F, G, and SH proteins, mumpsvirus HN and F proteins, human papilloma virus Ll protein, type 1 ortype 2 human immunodeficiency virus gp160 protein, herpes simplex virusand cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gMproteins, rabies virus G protein, Epstein Barr Virus gp350 protein,Filovirus G protein, bunyavirus G protein, flavivirus pre E, and NS1proteins, human metapneuomovirus (HMPV) G and F proteins, Ebola virus GPprotein, alphavirus E protein, and SARS and MERS S protein, andantigenic domains, fragments and epitopes thereof. Exemplary methods ofinserting one or more heterologous genes or transcriptional units into aparamyxovirus viral genome or antigenome are described in WO04/027037and US2013/0052718, each of which is incorporated by reference herein.In several embodiments, the heterologous gene included in therecombinant paramyxovirus genome encodes the ectodomain of a RSV Fprotein, such as a bovine RSV F protein or a human RSV F protein. HumanRSV can be classified into two groups: A and B. Groups A and B includesubgroups Al, A2, B1, and B2, based mainly on sequence variability ofthe attachment (G) and fusion (F) proteins. The RSV F ectodomain can bederived from any RSV group (such as Group A or Group B) or subgroup ofRSV, such as subgroup A1, A2, B1, or B2.

Exemplary human RSV F protein sequence from subgroup A2 andcorresponding GenBank reference (which is incorporated by referenceherein in its entirety) are set forth below:

RSV F A2 HEK Protein Sequence:

(SEQ ID NO: 1) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

RSV F B1 HEK Protein Sequence, Accession No. AAB82436:

MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK (SEQ ID NO: 2, GenBank Accession No. AAB82436,incorporated by reference herein in its entirety)

RSV F B1 HEK Nucleic Acid Sequence:

auggagcugcugauccacagguuaagugcaaucuuccuaacucuugcuauuaaugcauuguaccucaccucaagucagaacauaacugaggaguuuuaccaaucgacauguagugcaguuagcagagguuauuuuagugcuuuaagaacagguugguauaccagugucauaacaauagaauuaaguaauauaaaagaaaccaaaugcaauggaacugacacuaaaguaaaacuuauaaaacaagaauuagauaaguauaagaaugcagugacagaauuacagcuacuuaugcaaaacacaccagcugccaacaaccgggccagaagagaagcaccacaguauaugaacuauacaaucaauaccacuaaaaaccuaaauguaucaauaagcaagaagaggaaacgaagauuucugggcuucuuguuagguguaggaucugcaauagcaagugguauagcuguauccaaaguucuacaccuugaaggagaagugaacaagaucaaaaaugcuuuguuaucuacaaacaaagcuguagucagucuaucaaauggggucaguguuuuaaccagcaaaguguuagaucucaagaauuacauaaauaaccaauuauuacccauaguaaaucaacagagcugucgcaucuccaacauugaaacaguuauagaauuccagcagaagaacagcagauuguuggaaaucaacagagaauucagugucaaugcagguguaacaacaccuuuaagcacuuacauguuaacaaacagugaguuacuaucauugaucaaugauaugccuauaacaaaugaucagaaaaaauuaaugucaagcaauguucagauaguaaggcaacaaaguuauucuaucaugucuauaauaaaggaagaaguccuugcauauguuguacagcuaccuaucuaugguguaauagauacaccuugcuggaaauuacacacaucaccucuaugcaccaccaacaucaaagaaggaucaaauauuuguuuaacaaggacugauagaggaugguauugugauaaugcaggaucaguauccuucuuuccacaggcugacacuuguaaaguacaguccaaucgaguauuuugugacacuaugaacaguuugacauuaccaagugaagucagccuuuguaacacugacauauucaauuccaaguaugacugcaaaauuaugacaucaaaaacagacauaagcagcucaguaauuacuucucuuggagcuauagugucaugcuaugguaaaacuaaaugcacugcauccaacaaaaaucgugggauuauaaagacauuuucuaaugguugugacuaugugucaaacaaaggaguagauacugugucagugggcaacacuuuauacuauguaaacaagcuggaaggcaagaaccuuuauguaaaaggggaaccuauaauaaauuacuaugacccucuaguguuuccuucugaugaguuugaugcaucaauaucucaagucaaugaaaaaaucaaucaaaguuuagcuuuuauucguagaucugaugaauuacuacauaauguaaauacuggcaaaucuacuacaaauauuaugauaacuacaauuauuauaguaaucauuguaguauuguuaucauuaauagcuauugguuugcuguuguauugcaaagccaaaaacacaccaguuacacuaagcaaagaccaacuaaguggaaucaauaauauugcauucagcaaauag (SEQ ID NO: 3, GenBank Accession No.:AF013254.1, nucleotides 5666-7390, incorporated by reference herein inits entirety)

As illustrated by the sequences above, the hRSV F protein exhibitsremarkable sequence conservation, with sequence identity of more than85% across hRSV subgroups. In view of the conservation and breadth ofknowledge of RSV F sequences, the person of ordinary skill in the artcan easily identify corresponding RSV F amino acid positions betweendifferent RSV F strains and subgroups. The numbering of amino acidsubstitutions disclosed herein is made with reference to the exemplaryhRSV F protein sequence from the A2 stain set forth as SEQ ID NO: 1,unless context indicates otherwise.

For illustration purposes, the signal peptide, F2 polypeptide, pep27,F₁, F₁ ectodomain, transmembrane domain, and cytosolic domain of the RSVF protein from an A2 strain (SEQ ID NO: 1), are set forth as follows:

Signal peptide (SEQ ID NO: 1, residues 1-22): MELLILKANAITTILTAVTFCF F₂polypeptide (SEQ ID NO: 1, residues 23-109):ASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARR Pep27 (SEQ ID NO: 1, residues110-136): ELPRFMNYTLNNAKKTNVTLSKKRKRR F₁ (SEQ ID NO: 1, residues137-574): FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN F₁ ectodomain of mature protein(SEQ ID NO: 1, residues 137-529):FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITT

F₁ Transmembrane domain (SEQ ID NO: 1, residues 530-550):IIIVIIVILLSLIAVGLLLYC

F₁ CT (SEQ ID NO: 1, residues 551-574): KARSTPVTLSKDQLSGINNIAFSN

In some embodiments, the heterologous gene included in the recombinantparamyxovirus genome encodes the ectodomain of a human RSF F protein,wherein the RSV F ectodomain comprises an amino acid sequence at least85% (such as at least 90%, or at least 95%) identical to the RSVectodomain of one of SEQ ID NOs: 1 (WT RSV F A), 2 (WT RSV F B), 12 (A2HEK), 14 (A2 HEK+DS), or 19 (A2 HEK+DS-Cav1), or comprises the aminoacid sequence of the RSV ectodomain of SEQ ID NO: 12, 14, or 19.

In some embodiments the recombinant paramyxovirus can include a genomeincluding a heterologous gene encoding a recombinant hRSV F protein thathas been codon-optimized for expression in a human cell. For example,the gene encoding the recombinant hRSV F protein can be codon-optimizedfor human expression using a GA, DNA2.0 (D2), or GenScript (GS)optimization algorithm (see Example 1). Non-limiting examples of nucleicacid sequences encoding the RSV F protein that have been codon-optimizedfor expression in a human cell are provided as follows:

GeneArt Optimized RSV F A2 HEK DNA Sequence:

(SEQ ID NO: 4) atggaactgctgatcctgaaggccaacgccatcacaacaatcctgaccgccgtgaccttctgcttcgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgtccaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagtccacccccgccaccaacaaccgggccagaagagaactgccccggttcatgaactacaccctcaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggcttcctgctgggcgtgggcagcgccattgcctctggcgtggccgtgtctaaggtgctgcacctggaaggcgaagtgaacaagatcaagagcgccctgctgtccacaaacaaggccgtggtgtccctgagcaacggcgtgtccgtgctgacctccaaggtgctggatctgaagaactacatcgacaagcagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgccggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaatgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgagcatcatcaaagaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccccctgtgcaccaccaacacaaaagagggcagcaacatctgcctgacccggaccgaccggggctggtactgcgataatgccggcagcgtgtcattctttccacaggccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaacctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgaccagcaagaccgacgtgtccagctccgtgatcacctccctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtccgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgacgccagcatcagccaggtcaacgagaagatcaaccagagcctggccttcatcagaaagagcgacgagctgctgcacaatgtgaatgccggcaagagcaccacaaacatcatgatcaccactatcatcatcgtgatcatcgtcatcctgctgagtctgatcgccgtgggcctgctgctgtactgcaaggccagatccacccctgtgaccctgtccaaggatcagctgtccggcatcaacaatatcgccttctccaactga

GenScript Optimized RSV F A2 HEK DNA Sequence:

(SEQ ID NO: 5) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctctaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgacctctaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcgtcaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtctatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcatcatcgtgattatcgtcattctgctgtcactgatcgctgtggggctgctgctgtactgtaaggcaagaagcaccccagtcactctgtcaaaagaccagctgtcagggattaacaacattgccttcagtaactga

Additional examples of codon-optimized (for human expression) sequencesare provided below.

The RSV F protein encoded by the heterologous gene can include one ormore amino acid substitutions that improve expression of the RSV Fprotein, availability of the RSV F protein on the virion envelope, orstability of the RSV F protein, for example, in a prefusionconformation. In some embodiments, the RSV F protein can include aglutamic acid substitution at position 66, a proline substitution atposition 101, or both. For example the RSV F protein can include the“HEK” substitutions of a K66E substitution and a Q101P substitution.Exemplary DNA and protein sequences for a RSV F protein from the A2subgroup including the HEK amino acid substitutions are set forth below.

RSV F A2 Protein with HEK Substitutions (RSV F_A2_HEK): SEQ ID NO: 1GeneArt Optimized RSV F_A2_HEK DNA Sequence:

(SEQ ID NO: 6) atggaactgctgatcctgaaggccaacgccatcacaacaatcctgaccgccgtgaccttctgcttcgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgtccaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagtccacccccgccaccaacaaccgggccagaagagaactgccccggttcatgaactacaccctcaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggcttcctgctgggcgtgggcagcgccattgcctctggcgtggccgtgtctaaggtgctgcacctggaaggcgaagtgaacaagatcaagagcgccctgctgtccacaaacaaggccgtggtgtccctgagcaacggcgtgtccgtgctgacctccaaggtgctggatctgaagaactacatcgacaagcagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgccggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaatgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgagcatcatcaaagaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccccctgtgcaccaccaacacaaaagagggcagcaacatctgcctgacccggaccgaccggggctggtactgcgataatgccggcagcgtgtcattctttccacaggccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaacctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgaccagcaagaccgacgtgtccagctccgtgatcacctccctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtccgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgacgccagcatcagccaggtcaacgagaagatcaaccagagcctggccttcatcagaaagagcgacgagctgctgcacaatgtgaatgccggcaagagcaccacaaacatcatgatcaccactatcatcatcgtgatcatcgtcatcctgctgagtctgatcgccgtgggcctgctgctgtactgcaaggccagatccacccctgtgaccctgtccaaggatcagctgtccggcatcaacaatatcgccttctccaactgaGenScript Optimized RSV F_A2_HEK DNA Sequence:

(SEQ ID NO: 7) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctctaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgacctctaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcgtcaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtctatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcatcatcgtgattatcgtcattctgctgtcactgatcgctgtggggctgctgctgtactgtaaggcaagaagcaccccagtcactctgtcaaaagaccagctgtcagggattaacaacattgccttcagtaactga

In additional embodiments, the RSV F protein can include one or moreamino acid substitutions that stabilize the ectodomain of the RSV Fprotein in a prefusion conformation. For example, the RSV F protein caninclude the “DS” substitution of a pair of cysteine substitutions atpositions 155 and 290 that form a non-natural disulfide bond tostabilize the RSV F protein in its prefusion conformation. In someembodiments, the RSV F protein can include one or more cavity fillingamino acid substitutions at positions 190 and/or 207 to stabilize theprotein in a prefusion conformation. For example, the RSV F protein caninclude a 190F substitution and/or a 207L substitution. In someembodiments, the RSV F protein can include the “Cav1” substitutions ofS190F and a F207L. In some embodiments, the RSV F protein can includethe DS-Cav1 substitutions of S155C, S290C, S190F, and V207L to stabilizethe protein in a prefusion conformation. Exemplary DNA and proteinsequences for an RSV F protein (with a chimeric TM and/or CT domain)from the A2 subgroup including the DS-Cav1 amino acid substitutions areset forth as SEQ ID NOs: 10-11 and 21-23.

Additional amino acid substitutions and protein modifications that canbe used to stabilize the RSV F ectodomain in a prefusion conformationare disclosed, for example, in WO2014160463, which is incorporated byreference in its entirety. The HEK substitutions can be combined withany of the amino acid substitutions for stabilizing the RSV F protein ina prefusion conformation.

In several embodiments, the heterologous gene included in therecombinant paramyxovirus genome encodes a recombinant RSV F ectodomainlinked to a TM and CT of the F protein of the recombinant paramyxovirus.

In an embodiment, the recombinant paramyxovirus is a recombinant HPIV1including a recombinant HPIV1 genome including a heterologous geneencoding a recombinant hRSV F ectodomain. The RSV F ectodomain can belinked to a TM and CT from HPIV1 F protein, for example as set forth asresidues 1-23 of SEQ ID NO 31 (TM), residues 24-59 of SEQ ID NO: 31(CT), or SEQ ID NO: 31 (TM+CT). Exemplary sequences are provided below:

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and HPIV1 F CT Domain (RSV F A2_HEK_DS-Cav1_H1CT):

(SEQ ID NO: 133) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCRVRRLLVMINSTHNSPVNAYTLESRMRNPYMGNNSNGenScript Optimized RSV F A2_HEK_DS-Cav1_H1CT DNA Sequence:

(SEQ ID NO: 134) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgaccttcaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcctgaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcatcatcgtgattatcgtcattctgctgtcactgatcgctgtggggctgctgctgtactgtcgagtgcggagactgctggtcatgattaacagcacccacaattcccccgtcaacgcctacacactggagtctaggatgcgcaatccttatatggggaaca atagcaactgataghRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and HPIV1 F TM and CT Domains (RSV FA2_HEK_DS-Cav1_H1TMCT):

(SEQ ID NO: 135) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTQIIMIIIVCILIIIICGILYYLYRVRRLLVMINSTHNSPVNAYTLESRMRNPYMGNNSNGenScript Optimized RSV F A2_HEK_DS-Cav1_H1TMCT DNA Sequence:

(SEQ ID NO: 136) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgaccttcaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcctgaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacacagatcattatgatcattatcgtgtgcattctgattatcattatctgtggcatcctgtactatctgtaccgagtgcggagactgctggtcatgattaacagcacccacaattcccccgtcaacgcctacacactggagtctaggatgcgcaatccttatatgg ggaacaatagcaactgatag

In an embodiment, the recombinant paramyxovirus is a recombinant HPIV2including a recombinant HPIV2 genome including a heterologous geneencoding a recombinant hRSV F ectodomain. The RSV F ectodomain can belinked to a TM and CT from a HPIV2 F protein, for example as set forthas residues 1-28 of SEQ ID NO: 39 (TM), residues 29-66 of SEQ ID NO: 39(CT), or SEQ ID NO: 39 (TM+CT).

In an embodiment, the recombinant paramyxovirus can be a recombinantHPIV3 including a genome including a heterologous gene encoding arecombinant hRSV F ectodomain. The recombinant RSV F ectodomain can belinked to a TM and CT from a HPIV3 F protein, for example as set forthas residues 1-23 of SEQ ID NO 46 (TM), residues 24-46 of SEQ ID NO: 46(CT), or SEQ ID NO: 46 (TM+CT). Exemplary sequences are provided below:

hRSV F p protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and HPIV3 F CT Domain (RSV F_HEK_DS-Cav1_H3CT) ProteinSequence:

(SEQ ID NO: 8) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKYYRIQKRNRVDQNDKPYVLTNK

GenScript Optimized RSV F_HEK_DS-Cav1_H3CT DNA Sequence:

(SEQ ID NO: 9) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgaccttcaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcctgaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcatcatcgtgattatcgtcattctgctgtcactgatcgctgtggggctgctgctgtactgtaagtactaccgtatccagaagaggaacagagttgaccagaacgataagccatacgtgctcactaacaagtga

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and HPIV3 F TM and CT Domains (RSV F_HEK_DS-Cav1_H3TMCT)Protein Sequence:

(SEQ ID NO: 10) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK

GenScript Optimized RSV F_HEK_DS-Cav1_H3TMCT DNA Sequence:

(SEQ ID NO: 11) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgaccttcaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcctgaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcatcatcatcttgatcatgatcatcatcctgttcatcatcaacatcacaatcatcaccatcgctatcaagtactaccgtatccagaagaggaacagagttgaccagaacgataagccatacgtgctcactaacaagtga

In an embodiment, the recombinant paramyxovirus is a chimeric PIVincluding a recombinant viral genome encoding HPIV3 F and HN proteinsand BPIV3 N, P, C, V, M, and L proteins, wherein the viral genomefurther includes a heterologous gene encoding a recombinant hRSV Fectodomain linked to a TM and/or CT from a BPIV3 F protein, for exampleas set forth as residues 1-21 of SEQ ID NO 53 (TM), residues 22-57 ofSEQ ID NO: 53 (CT) or SEQ ID NO: 53 (TM+CT). Exemplary DNA and proteinsequences for recombinant RSV F proteins of the A2 subgroup that includethe HEK, DS, and/or Cav1 substitutions, as well as a heterologous TMand/or CT domains from BPIV3 F protein that can be used in a disclosedrecombinant paramyxovirus are set forth below.

hRSV F Protein from an A2 Strain Including HEK Substitutions, and BPIV3F TM and CT Domains (RSV F_A2_HEK_B3TMCT) Protein Sequence:

(SEQ ID NO: 12) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ

GeneArt Optimized RSV F_A2_HEK_B3TMCT DNA Sequence:

(SEQ ID NO: 13) atggaactgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgctttgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgagcaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaatgccgtgaccgaactgcagctgctgatgcagagcacccccgccaccaacaaccgggccagaagagaactgcccagattcatgaactacaccctgaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggctttctgctgggagtgggaagcgccattgctagcggagtggccgtgtctaaggtgctgcacctggaaggcgaagtgaacaagatcaagtccgccctgctgagcaccaacaaggccgtggtgtctctgagcaacggcgtgtccgtgctgaccagcaaggtgctggatctgaagaactacatcgacaaacagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgctggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaacgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgagcattatcaaagaagaggtgctggcctacgtggtgcagctgcctctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccctctgtgcaccaccaacaccaaagagggctccaacatctgcctgacccggaccgacagaggctggtactgcgataatgccggctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctccaagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcaccatcatcattgtgatgattatcatcctcgtgatcatcaacatcacaatcatcgtcgtgattattaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga

hRSV F Protein from an A2 Strain Including HEK and DS Substitutions,hRSV F TM Domain and BPIV3 F CT Domain (RSV F_A2_HEK_DS_B3CT) ProteinSequence:

(SEQ ID NO: 14) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCIIKFHRIQGKDQNDKNSEPYILTNRQ

GeneArt Optimized RSV F_A2_HEK_DS_B3CT DNA Sequence:

(SEQ ID NO: 15) atggaactgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgctttgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgagcaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaatgccgtgaccgaactgcagctgctgatgcagagcacccccgccaccaacaaccgggccagaagagaactgcccagattcatgaactacaccctgaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggctttctgctgggagtgggaagcgccattgctagcggagtggccgtgtgcaaggtgctgcacctggaaggcgaagtgaacaagatcaagtccgccctgctgagcaccaacaaggccgtggtgtctctgagcaacggcgtgtccgtgctgaccagcaaggtgctggatctgaagaactacatcgacaaacagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgctggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaacgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgtgcatcatcaaagaagaggtgctggcctacgtggtgcagctgcctctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccctctgtgcaccaccaacaccaaagagggctccaacatctgcctgacccggaccgacagaggctggtactgcgataatgccggctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctccaagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcatcatcgtgattatcgtgatcctgctgagcctgatcgccgtgggcctgctgctgtactgtatcatcaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, hRSV F TM Domain and BPIV3 F CT Domain (RSVF_A2_HEK_DS-Cav1_B3CT) Protein Sequence:

(SEQ ID NO: 16) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCIIKFHRIQGKDQNDKNSEPYILTNRQ

GeneArt Optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA Sequence:

(SEQ ID NO: 17) atggaactgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgctttgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgagcaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaatgccgtgaccgaactgcagctgctgatgcagagcacccccgccaccaacaaccgggccagaagagaactgcccagattcatgaactacaccctgaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggctttctgctgggagtgggaagcgccattgctagcggagtggccgtgtgcaaggtgctgcacctggaaggcgaagtgaacaagatcaagtccgccctgctgagcaccaacaaggccgtggtgtctctgagcaacggcgtgtccgtgctgaccttcaaggtgctggatctgaagaactacatcgacaaacagctgctgcccatcttgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgctggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaacgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgtgcatcatcaaagaagaggtgctggcctacgtggtgcagctgcctctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccctctgtgcaccaccaacaccaaagagggctccaacatctgcctgacccggaccgacagaggctggtactgcgataatgccggctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctccaagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcatcatcgtgattatcgtgatcctgctgagcctgatcgccgtgggcctgctgctgtactgtatcatcaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga

GenScript Optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA Sequence:

(SEQ ID NO: 18) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgaccttcaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcctgaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcatcatcgtgattatcgtcattctgctgtcactgatcgctgtggggctgctgctgtactgtatcattaagttccaccggatccagggcaaggaccagaacgataaaaatagcgagccctacattctgaccaacagacag

hRSV F Protein from an A2 Strain Including HEK and DS Substitutions, andBPIV3 F TM and CT Domains (RSV F_A2_HEK_DS_B3TMCT) Protein Sequence:

(SEQ ID NO: 19) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ

GeneArt Optimized RSV F_A2_HEK_DS_B3TMCT DNA Sequence:

(SEQ ID NO: 20) atggaactgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgctttgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgagcaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaatgccgtgaccgaactgcagctgctgatgcagagcacccccgccaccaacaaccgggccagaagagaactgcccagattcatgaactacaccctgaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggctttctgctgggagtgggaagcgccattgctagcggagtggccgtgtgcaaggtgctgcacctggaaggcgaagtgaacaagatcaagtccgccctgctgagcaccaacaaggccgtggtgtctctgagcaacggcgtgtccgtgctgaccagcaaggtgctggatctgaagaactacatcgacaaacagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgctggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaacgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgtgcatcatcaaagaagaggtgctggcctacgtggtgcagctgcctctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccctctgtgcaccaccaacaccaaagagggctccaacatctgcctgacccggaccgacagaggctggtactgcgataatgccggctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctccaagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcaccatcatcattgtgatgattatcatcctcgtgatcatcaacatcacaatcatcgtcgtgattattaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga

Genescript Optimized RSV F_A2_HEK_DS_B3TMCT DNA Sequence:

(SEQ ID NO: 137) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgacctccaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcgtcaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcaccatcattatcgtgatgattatcattctggtcatcattaacatcacaatcattgtggtcatcattaagttccaccggattcagggcaaggaccagaacgataaaaatagcgagccctacatcctgaccaatagacagtga

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and BPIV3 F TM and CT domains (RSVF_A2_HEK_DS-Cav1_B3TMCT) Protein Sequence:

(SEQ ID NO: 21) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ

GeneArt Optimized RSV F_A2_HEK_DS-Cav1_B3TMCT DNA Sequence:

(SEQ ID NO: 22) atggaactgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgctttgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgagcaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaatgccgtgaccgaactgcagctgctgatgcagagcacccccgccaccaacaaccgggccagaagagaactgcccagattcatgaactacaccctgaacaacgccaaaaagaccaacgtgaccctgagcaagaagcggaagcggcggttcctgggctttctgctgggagtgggaagcgccattgctagcggagtggccgtgtgcaaggtgctgcacctggaaggcgaagtgaacaagatcaagtccgccctgctgagcaccaacaaggccgtggtgtctctgagcaacggcgtgtccgtgctgaccttcaaggtgctggatctgaagaactacatcgacaaacagctgctgcccatcttgaacaagcagagctgcagcatcagcaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctggaaatcacccgcgagttcagcgtgaacgctggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaacgacatgcccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgtgcatcatcaaagaagaggtgctggcctacgtggtgcagctgcctctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccctctgtgcaccaccaacaccaaagagggctccaacatctgcctgacccggaccgacagaggctggtactgcgataatgccggctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctccaagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcaccatcatcattgtgatgattatcatcctcgtgatcatcaacatcacaatcatcgtcgtgattattaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga

GenScript Optimized RSV F_A2_HEK_DS-Cav1_B3TMCT DNA Sequence:

(SEQ ID NO: 23) atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgtcctgaccttcaaggtgctggacctgaaaaattatattgataagcagctgctgcctatcctgaacaaacagagctgctccatttctaatatcgagacagtgatcgaattccagcagaagaacaatagactgctggagattaccagagagttcagcgtgaacgccggcgtcaccacacccgtgtccacctacatgctgacaaatagtgagctgctgtcactgattaacgacatgcctatcaccaatgatcagaagaaactgatgtccaacaatgtgcagatcgtcagacagcagagttactcaatcatgtgcatcattaaggaggaagtcctggcctacgtggtccagctgccactgtatggcgtgatcgacaccccctgctggaaactgcatacatctcctctgtgcactaccaacacaaaggaaggaagtaatatctgcctgactcgaaccgaccggggatggtactgtgataacgcaggcagcgtgtccttctttccacaggccgagacctgcaaggtccagagcaacagggtgttctgtgacactatgaatagcctgaccctgccttccgaagtcaacctgtgcaatgtggacatctttaatccaaagtacgattgtaagatcatgactagcaagaccgatgtcagctcctctgtgattacttctctgggggccatcgtgagttgctacggaaagacaaaatgtactgccagcaacaaaaatcgcggcatcattaagaccttctccaacgggtgcgactatgtctctaacaagggcgtggatacagtgagtgtcgggaacactctgtactatgtcaataagcaggagggaaaaagcctgtacgtgaagggcgaacccatcattaacttctatgaccccctggtgttcccttccgacgagtttgatgcatctattagtcaggtgaacgaaaaaatcaatcagagtctggcctttattcggaagtcagatgagctgctgcacaacgtgaatgctggcaaatctacaactaacatcatgatcaccacaatcaccatcattatcgtgatgattatcattctggtcatcattaacatcacaatcattgtggtcatcattaagttccaccggattcagggcaaggaccagaacgataaaaatagcgagccctacatcctgaccaatagacagtga

In an embodiment, the recombinant paramyxovirus includes a recombinantSendai virus genome including a heterologous gene encoding a recombinanthRSV F ectodomain. In such embodiments, the TM and CT linked to the RSVF ectodomain can be from a Sendai virus F protein, for example as setforth as residues 1-21 of SEQ ID NO: 103 (TM), residues 22-65 of SEQ IDNO: 103 (CT), or SEQ ID NO: 103 (TM+CT). For example, in someembodiments, the recombinant hRSV F ectodomain linked to the Sendaivirus TM and/or CT can include the amino acid sequence set forth as oneof SEQ ID NOs: 105-108:

hRSV F Protein from an A2 Strain Including HEK Substitutions, and SendaiVirus F CT Domain (RSV F_A2_HEK_SeVCT) Protein Sequence.

(SEQ ID NO: 105) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR

hRSV F Protein from an A2 Strain Including HEK Substitutions, and SendaiVirus F TM and CT Domains (RSV F_A2_HEK_SeVTMCT) Protein Sequence.

(SEQ ID NO: 106) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTVITIIVVMVVILVVIIVIIIVLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and Sendai Virus F CT Domains (RSV F_A2_HEK_SeVCT)Protein Sequence

(SEQ ID NO: 107) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and Sendai Virus F TM and CT Domains (RSVF_A2_HEK_SeVTMCT) Protein Sequence

(SEQ ID NO: 108) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTVITIIVVMVVILVVIIVIIIVLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR

In an embodiment, the recombinant paramyxovirus includes a recombinantNDV genome including a heterologous gene encoding a recombinant hRSV Fectodomain. In such embodiments, the TM and CT linked to the RSV Fectodomain can be from a NDV virus F protein, cytoplasmic tail, forexample as set forth as residues 1-21 of SEQ ID NO: 104 (TM), residues22-49 of SEQ ID NO: 104 (CT), or SEQ ID NO: 104 (TM+CT). For example, insome embodiments, the recombinant hRSV F ectodomain linked to the NDV TMand/or CT can include the amino acid sequence set forth as one of SEQ IDNOs: 109-113:

hRSV F Protein from an A2 Strain Including HEK Substitutions, and NDV FCT Domains (RSV F_A2_HEK_NDVCT) Protein Sequence

(SEQ ID NO: 109) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCMYKQKAQQKTLLWLGNNTLDQMRATTKM

hRSV F Protein from an A2 Strain Including HEK Substitutions, and NDV FTM and CT Domains (RSV F_A2_HEK_NDVTMCT) Protein Sequence

(SEQ ID NO: 110) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIVLTIISLVFGILSLILACYLMYKQKAQQKTLLWLGNNTLDQMRATTKM

hRSV F Protein from an A2 Strain Including HEK and DS-Cav1Substitutions, and NDV F CT Domains (RSV F_A2_HEK_NDVCT) ProteinSequence

(SEQ ID NO: 112) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCMYKQKAQQKTLLWLGNNTLDQMRATTKM

hRSV F Pprotein from an A2 Strain Including HEK and DS-Cav1Substitutions, and NDV F TM and CT Domains (RSV F_A2_HEK_NDVTMCT)Protein Sequence

(SEQ ID NO: 113) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTVITIIVVMVVILVVIIVIIIVLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKRH. Additional Description of Recombinant Paramyxovirus

Particular Embodiments

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant parainfluenza virus (PIV) comprising a viral genomecomprising, from upstream to downstream, a PIV genomic promoter followedby PIV N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and207L substitutions and is linked to a TM and CT of the PIV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and islinked to a TM and CT of the HPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a TM and CT of theHPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to aTM and CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant

HPIV3 comprising a viral genome comprising, from upstream to downstream,a HPIV3 genomic promoter followed by HPIV3 N, P, M, F, HN, and L genes,and further comprising a heterologous gene encoding a type I membraneprotein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a TM and CT of theHPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and islinked to a TM and CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C, 190F, and 207L substitutions and is linked to a TM and CT ofthe BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and islinked to a TM and CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C, 190F, and 207L substitutions and is linked to a TM and CT ofthe BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant

RSV F ectodomain, wherein the heterologous gene is located between thegenomic promoter and the gene encoding the N protein, and wherein theRSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207Lsubstitutions and is linked to a TM and CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207Lsubstitutions and is linked to a TM and CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a TM and CT of theHPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207Lsubstitutions and is linked to a TM and CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and207L substitutions and is linked to a TM and CT of the sendai virus Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L gene, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutionsand is linked to a TM and CT of the sendai virus F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgenes, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genomic promoter and the geneencoding the N protein, and wherein the RSV F ectodomain comprises 66E,101P, 155C, 290C, 190F, and 207L substitutions and is linked to a TM andCT of the NDV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a TM and CT of theNDV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to aTM and CT of the PIV5 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L gene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a TM and CT of thePIV5 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant parainfluenza virus (PIV) comprising a viral genomecomprising, from upstream to downstream, a PIV genomic promoter followedby PIV N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and207L substitutions and is linked to a CT of the PIV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and islinked to a CT of the HPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a CT of the HPIV1 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to aCT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C, 190F, and 207L substitutions and is linked to a CT of theHPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and islinked to a CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C, 190F, and 207L substitutions and is linked to a CT of theBPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant

BPIV3 comprising a viral genome comprising, from upstream to downstream,a BPIV3 genomic promoter followed by BPIV3 N, P, M, F, HN, and L genes,and further comprising a heterologous gene encoding a type I membraneprotein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genomic promoter and the geneencoding the N protein, and wherein the RSV F ectodomain comprises 66E,101P, 155C, 290C, 190F, and 207L substitutions and is linked to a CT ofthe BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C, 190F, and 207L substitutions and is linked to a CT of theBPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a CT of the BPIV3 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207Lsubstitutions and is linked to a CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a CT of the HPIV3 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207Lsubstitutions and is linked to a CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and207L substitutions and is linked to a CT of the sendai virus F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L gene, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutionsand is linked to a CT of the sendai virus F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgenes, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genomic promoter and the geneencoding the N protein, and wherein the RSV F ectodomain comprises 66E,101P, 155C, 290C, 190F, and 207L substitutions and is linked to a CT ofthe NDV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a CT of the NDV Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to aCT of the PIV5 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L gene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C, 190F, and 207L substitutions and is linked to a CT of the PIV5 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant parainfluenza virus (PIV) comprising a viral genomecomprising, from upstream to downstream, a PIV genomic promoter followedby PIV N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290Csubstitutions and is linked to a TM and CT of the PIV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a TM andCT of the HPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a TM and CT of the HPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C substitutions and is linked to a TM and CT of theHPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C substitutions and is linked to a TM and CT of the HPIV3 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a TM and

CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C substitutions and is linked to a TM and CT of the BPIV3 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a TM andCT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C substitutions and is linked to a TM and CT of the BPIV3 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a TM and CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C substitutions andis linked to a TM and CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a TM and CT of the HPIV3 F protein.In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant

B/HPIV3 comprising a viral genome comprising, from upstream todownstream, a BPIV3 genomic promoter followed by BPIV3 N, P, and Mgenes, HPIV3 F and HN genes, and a BPIV3 L gene, and further comprisinga heterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain comprises 66E, 101P, 155C, 290C substitutions and is linkedto a TM and CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290Csubstitutions and is linked to a TM and CT of the sendai virus Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L gene, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain comprises 66E, 101P, 155C, 290C substitutions and is linkedto a TM and CT of the sendai virus F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgenes, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genomic promoter and the geneencoding the N protein, and wherein the RSV F ectodomain comprises 66E,101P, 155C, 290C substitutions and is linked to a TM and CT of the NDV Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a TM and CT of the NDV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C substitutions and is linked to a TM and CT of thePIV5 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L gene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a TM and CT of the PIV5 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant parainfluenza virus (PIV) comprising a viral genomecomprising, from upstream to downstream, a PIV genomic promoter followedby PIV N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290Csubstitutions and is linked to a CT of the PIV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a CT ofthe HPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV1 comprising a viral genome comprising, from upstreamto downstream, a HPIV1 genomic promoter followed by HPIV1N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a CT of the HPIV1 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C substitutions and is linked to a CT of the HPIV3 Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C substitutions and is linked to a CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a CT ofthe BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant HPIV3 comprising a viral genome comprising, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C substitutions and is linked to a CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a CT ofthe BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant BPIV3 comprising a viral genome comprising, from upstreamto downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F,HN, and L genes, and further comprising a heterologous gene encoding atype I membrane protein comprising a recombinant RSV F ectodomain,wherein the heterologous gene is located between the genes encoding theN and P proteins, and wherein the RSV F ectodomain comprises 66E, 101P,155C, 290C substitutions and is linked to a CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C substitutions andis linked to a CT of the BPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genomic promoter and the gene encoding the Nprotein, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant B/HPIV3 comprising a viral genome comprising, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and furthercomprising a heterologous gene encoding a type I membrane proteincomprising a recombinant RSV F ectodomain, wherein the heterologous geneis located between the genes encoding the N and P proteins, and whereinthe RSV F ectodomain comprises 66E, 101P, 155C, 290C substitutions andis linked to a CT of the HPIV3 F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L genes, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain comprises 66E, 101P, 155C, 290Csubstitutions and is linked to a CT of the sendai virus F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant sendai virus comprising a viral genome comprising, fromupstream to downstream, a sendai virus genomic promoter followed bysendai virus N, P, M, F, HN, and L gene, and further comprising aheterologous gene encoding a type I membrane protein comprising arecombinant RSV F ectodomain, wherein the heterologous gene is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain comprises 66E, 101P, 155C, 290C substitutions and is linkedto a CT of the sendai virus F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgenes, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genomic promoter and the geneencoding the N protein, and wherein the RSV F ectodomain comprises 66E,101P, 155C, 290C substitutions and is linked to a CT of the NDV Fprotein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant NDV comprising a viral genome comprising, from upstream todownstream, a NDV genomic promoter followed by NDV N, P, M, F, HN, and Lgene, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant RSV F ectodomain, wherein theheterologous gene is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain comprises 66E, 101P, 155C,290C substitutions and is linked to a CT of the NDV F protein.

In some embodiments, a recombinant paramyxovirus is provided, comprisinga recombinant PIV5 comprising a viral genome comprising, from upstreamto downstream, a PIV5 genomic promoter followed by PIV5 N, P, M, F, HN,and L genes, and further comprising a heterologous gene encoding a typeI membrane protein comprising a recombinant RSV F ectodomain, whereinthe heterologous gene is located between the genomic promoter and thegene encoding the N protein, and wherein the RSV F ectodomain comprises66E, 101P, 155C, 290C substitutions and is linked to a CT of the PIV5 Fprotein. In some embodiments, a recombinant paramyxovirus is provided,comprising a recombinant PIV5 comprising a viral genome comprising, fromupstream to downstream, a PIV5 genomic promoter followed by PIV5 N, P,M, F, HN, and L gene, and further comprising a heterologous geneencoding a type I membrane protein comprising a recombinant RSV Fectodomain, wherein the heterologous gene is located between the genesencoding the N and P proteins, and wherein the RSV F ectodomaincomprises 66E, 101P, 155C, 290C substitutions and is linked to a CT ofthe PIV5 F protein.

In any of the embodiments of a recombinant paramyxovirus disclosedherein that includes a viral genome including a heterologous geneencoding an RSV F ectodomain (such as any of the recombinantparamyxoviruses discussed above, the heterologous gene encoding therecombinant RSV F ectodomain can encodes a polypeptide sequencecomprising RSV F positions 1-529.

Additional Description

The disclosed recombinant paramyxoviruses are self-replicating, that isthey are capable of replicating following infection of an appropriatehost cell. In several embodiments, the recombinant paramyxoviruses havean attenuated phenotype, for example when administered to a humansubject.

Attenuation of the recombinant paramyxoviruses can be achieved usingvarious methods known in the art, for example, by introduction of one ormore mutations that cause a change in the biological function of therecombinant paramyxoviruses result in the attenuated phenotype.Insertion of the heterologous gene can also result in an attenuatedphenotype. Preferably, the paramyxovirus comprising a genome encoding aheterologous gene is attenuated about 100 to 5000 fold or more in a cellor mammal compared to wild type paramyxovirus.

The disclosed recombinant paramyxoviruses can be tested in well-knownand in vitro and in vivo models to confirm adequate attenuation,resistance to phenotypic reversion, and immunogenicity. In in vitroassays, the modified paramyxovirus can be tested for one or more desiredphenotypes, such as, for example, temperature sensitive replication. Thedisclosed recombinant paramyxoviruses can also be tested in animalmodels of infection with PIV and/or the viral pathogen of theheterologous gene included in the recombinant virus (e.g., RSV). Avariety of animal models are known.

The recombinant attenuated paramyxoviruses are preferably attenuatedabout 100 to 5000 fold in a cell or mammal compared to wild typeparamyxovirus. In some embodiments, it is preferred that the level ofviral replication in vitro is sufficient to provide for production ofviral vaccine for use on a wide spread scale. In some embodiments, it ispreferred that the level of viral replication of attenuatedparamyxovirus in vitro is at least 10⁶, more preferably at least 10′,and most preferably at least 10⁸ per ml. The attenuating mutation ispreferably one that is stable. A recombinant paramyxovirus with at leasttwo, three, four or ever more attenuating mutations is likely to be morestable.

Ongoing preclinical studies have identified a number of mutations ormodifications that are attenuating for HPIV1, HPIV2, and HPIV3, andwhich can be introduced by reverse genetics to produce attenuatedstrains as potential vaccines and vector backbones. The inclusion of aforeign gene into an HPIV backbone also is attenuating on its own. Thismay due to a variety of effects including the increase in genome lengthand gene number as well as the effects of the foreign protein. Whateverthe cause, the attenuating effect of the insert also has to be takeninto account when attempting to achieve the appropriate level ofattenuation.

Attenuated strains of HPIV1, 2, and 3 have been in or are presently inclinical studies in seronegative infants and children (Karron, et al.2012. Vaccine 30:3975-3981; Schmidt, et al. 2011. Expert Rev. Respir.Med. 5:515-526). These attenuated HPIV1, HPIV2, and HPIV3 strains, orversions thereof, are potential vectors for expressing the heterologousRSV F protein.

Examples of modifications to the genome of a paramyxovirus that providefor an attenuated phenotype are known in the art and have beendescribed, for example, in US Patent Publications 2012/0045471;2010/0119547; 2009/0263883; 2009/0017517; U.S. Pat. Nos. 8,084,037;6,410,023; 8,367,074; 7,951,383; 7,820,182; 7,704,509; 7,632,508;7,622,123; 7,250,171; 7,208,161; 7,201,907; 7,192,593; 2012/0064112;20140186397; and Newman et al. 2002. Virus genes 24:77-92, Tang et al.,2003. J Virol, 77(20):10819-10828; Basavarajappa et al. 2014 Vaccine,32: 3555-3563; McGinnes et al., J. Virol., 85: 366-377, 2011; and Joneset al., Vaccine, 30:959-968, 2012, each of which is incorporated byreference herein in its entirety. For example, attenuation of PIV3 canbe achieved by the presence of BPIV3-derived genes, which confers a hostrange restriction in primates including humans, such as the B/HPIV3virus that contains BPIV3 genes except for the F and HN from HPIV3(Skiadopoulos M H et al J Virol 77:1141-8, 2003). Sendai virus also isrestricted in primates due to a host range restruction (Jones B G et alVaccine 30:959-968 2012). Another means of attenuation is exemplified bymissense mutations that can occur in multiple genes, such as in the cp45HPIV3 virus (Skiadopoulos M H et al J Virol 73:1374-81 1999). Otherexamples of attenuating point mutations are provided for HPIV1 inExample 2, below. Deletion of one or several codons also can confer anattenuation phenotype, as exemplified by HPIV1 in Example 2. As alsoexemplified in Example 1, the presence of vector TM plus CT, or CTdomains linked to a heterologous ectodomain can strongly attenuate thevector. Other examples of attenuating mutations in HPIV1 are describedby Bartlett E J et al Virol J 4:67 2007), and for HPIV2 by Nolan S M etal, Vaccine 23:4765-4774 2005). The deletion of all or part of one ormore accessory genes also is a means of attenuation (Durbin A Virology261:319-330 1999).

Immunogenicity of a recombinant attenuated paramyxovirus can be assessedin an animal model (such as a non-human primate, for example an Africangreen monkey) by determining the number of animals that form antibodiesto the paramyxovirus after one immunization and after a secondimmunization, and by measuring the magnitude of that response. In someembodiments, a recombinant paramyxovirus has sufficient immunogenicityif about 60 to 80% of the animals develop antibodies after the firstimmunization and about 80 to 100% of the animals develop antibodiesafter the second immunization. Preferably, the immune response protectsagainst infection by both the originating paramyxovirus and the viralpathogen from which the heterologous gene included in the recombinantparamyxovirus is derived.

I. Additional Vectors

It will be appreciated that the recombinant RSV F proteins and nucleicacid molecules encoding same can be included (or expressed) on vectorsother than a PIV vector. For example, plasmid vectors, as well as otherviral vectors can be used, for example, for expression of therecombinant RSV F protein or fragment thereof in a host cell, or forimmunization of a subject as disclosed herein. In some embodiments, thevectors can be administered to a subject as part of a prime-boostvaccination. In several embodiments, the vectors are included in avaccine, such as a primer vaccine or a booster vaccine for use in aprime-boost vaccination.

In several examples, the vector can be a viral vector that isreplication-competent and/or attenuated. The viral vector also can beconditionally replication-competent. In other examples, the viral vectoris replication-deficient in host cells.

A number of viral vectors have been constructed, that can be used toexpress the recombinant RSV F protein or immunogenic fragment thereof,including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol.,73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol.,158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia etal., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl.Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian(Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouploset al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top.Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol.,5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann etal., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990,J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol.,66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.).

In several embodiments, the viral vector can include an adenoviralvector that expresses a disclosed recombinant RSV F protein orimmunogenic fragment thereof (such as the RSV F ectodomain). Adenovirusfrom various origins, subtypes, or mixture of subtypes can be used asthe source of the viral genome for the adenoviral vector. Non-humanadenovirus (e.g., simian, chimpanzee, gorilla, avian, canine, ovine, orbovine adenoviruses) can be used to generate the adenoviral vector. Forexample, a simian adenovirus can be used as the source of the viralgenome of the adenoviral vector. A simian adenovirus can be of serotype1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any othersimian adenoviral serotype. A simian adenovirus can be referred to byusing any suitable abbreviation known in the art, such as, for example,SV, SAdV, SAV or sAV. In some examples, a simian adenoviral vector is asimian adenoviral vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33,38, or 39. In one example, a chimpanzee serotype C Ad3 vector is used(see, e.g., Peruzzi et al., Vaccine, 27:1293-1300, 2009). Humanadenovirus can be used as the source of the viral genome for theadenoviral vector. Human adenovirus can be of various subgroups orserotypes. For instance, an adenovirus can be of subgroup A (e.g.,serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16,21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6),subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24,25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g.,serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassifiedserogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype.The person of ordinary skill in the art is familiar with replicationcompetent and deficient adenoviral vectors (including singly andmultiply replication deficient adenoviral vectors). Examples ofreplication-deficient adenoviral vectors, including multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and7,195,896, and International Patent Application Nos. WO 94/28152, WO95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO97/21826, and WO 03/02231 1.

III. Recombinant Methods, Vectors, and Host Cells

The recombinant paramyxoviruses and polynucloetides disclosed herein canbe produced by synthetic and recombinant methods. Accordingly,polynucleotides encoding infectious paramyxovirus clones and host cellsincluding the infectious clone, as well as methods of making suchvectors and host cells by recombinant methods are also provided.

Isolated nucleic acid molecules encoding any of the recombinant RSV Fproteins disclosed herein are also provided.

As discussed above, the disclosed paramyxovirus or polynucleotides maybe synthesized or prepared by techniques well known in the art. See, forexample, WO94/027037 and US20130052718. Nucleotide sequences for wildtype paramyxovirus genomes are known and readily available, for example,on the Internet at GenBank (accessible at ncbi-nlm-nihgov/entrez). Thenucleotide sequences encoding the disclosed recombinant paramyxovirusmay be synthesized or amplified using methods known to those of ordinaryskill in the art including utilizing DNA polymerases in a cell freeenvironment. Further, one of skill in the art can readily use thegenetic code to construct a variety of functionally equivalent nucleicacids, such as nucleic acids which differ in sequence but which encodethe same protein sequence.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, New York, 2012, and Ausubel etal. (In Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, through supplement 104, 2013). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAChemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.),Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis. ), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill.

The genome of the recombinant paramyxovirus can include one or morevariations (for example, mutations that cause an amino acid deletion,substitution, or insertion) as long as the resulting recombinantparamyxovirus retains the desired biological function, such as a levelof attenuation or immunogenicity. These variations in sequence can benaturally occurring variations or they can be engineered through the useof genetic engineering technique known to those skilled in the art.Examples of such techniques are found in see, e.g., Sambrook et al.(Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor,New York, 2012) and Ausubel et al. (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, through supplement 104, 2013, bothof which are incorporated herein by reference in their entirety.

Modifications can be made to a nucleic acid encoding described hereinwithout diminishing its biological activity Amino acid substitutions,insertions, and deletions can be made using known recombinant methodssuch as oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, PCR mutagenesis, site-directed mutagenesis, cassettemutagenesis, restriction selection mutagenesis, and the like (see, e.g.,Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^(th) ed, ColdSpring Harbor, New York, 2012, and Ausubel et al. (In Current Protocolsin Molecular Biology, John Wiley & Sons, New York, through supplement104, 2013). Some modifications can be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, termination codons, a methionine added at theamino terminus to provide an initiation, site, additional nucleotidesplaced on either terminus to create conveniently located restrictionsites.

“Conservative” amino acid substitutions are those substitutions that donot substantially affect or decrease a function of a protein, such asthe ability of the protein to induce an immune response whenadministered to a subject. The term conservative variation also includesthe use of a substituted amino acid in place of an unsubstituted parentamino acid. Furthermore, one of ordinary skill will recognize thatindividual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids (forinstance less than 5%, in some embodiments less than 1%) in an encodedsequence are conservative variations where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The disclosed recombinant paramyxovirus can be produced from virusisolated from biological samples. The polynucleotides and vectors may beproduced by standard recombinant methods known in the art, such aspolymerase chain reaction (Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4th ed, Cold Spring Harbor, New York, 2012, andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, through supplement 104, 2013). Methods of altering ormodifying nucleic acid sequences are also known to those of skill in theart.

The paramyxovirus genome may be assembled from polymerase chain reactioncassettes sequentially cloned into a vector including a selectablemarker for propagation in a host. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria.

The polynucleotide may be inserted into a replicable vector for cloningusing standard recombinant methods. Various vectors are publiclyavailable. The vector may, for example, be in the form of a plasmid,cosmid, viral particle, or phage. The appropriate nucleic acid sequencemay be inserted into the vector by a variety of procedures. In general,a nucleic acid is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors including one or more of these components employsstandard ligation techniques that are known to the skilled artisan.

Examples of suitable replicable vectors include, without limitation,pUC19 or pTM1. The polynucleotide can be operably linked to anappropriate promoter such as, for example, T7 polymerase promoter,cytomegalovirus promoter, cellular polymerase II promoter, or SP1promoter. The replicable vectors may further include sites fortranscription initiation, transcription termination, and a ribosomebinding site for translation.

Introduction of a recombinant vector composed of a paramyxovirus genomeor polynucleotide encoding a paramyxovirus protein into a host cell,such as for example a bacterial cell or eukaryotic cell, can be affectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, electricalnuclear transport, chemical transduction, electrotransduction,infection, or other methods. Such methods are described in standardlaboratory manuals such as Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4^(th) ed, Cold Spring Harbor, New York, 2012, andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, through supplement 104, 2013. Commercial transfectionreagents, such as Lipofectamine (Invitrogen, Carlsbad, Calif.) andFuGENE 6TM (Roche Diagnostics, Indianapolis, Ind.), are also available.Suitable host cells include, but are not limited to, HEp-2 cells,FRhL-DBS2 cells, LLC-MK2 cells, MRC-5 cells, and Vero cells.

IV. Immunogenic Compositions

Immunogenic compositions comprising a recombinant paramyxoviruses asdescribed herein (such as a recombinant PIV including a genome encodinga heterologous recombinant RSV F protein) and a pharmaceuticallyacceptable carrier are also provided. Such compositions can beadministered to subjects by a variety of administration modes known tothe person of ordinary skill in the art, for example, by an intranasalroute. Actual methods for preparing administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remingtons Pharmaceutical Sciences,19^(th) Ed., Mack Publishing Company, Easton, Pa. 1995.

Thus, a recombinant paramyxovirus described herein can be formulatedwith pharmaceutically acceptable carriers to help retain biologicalactivity while also promoting increased stability during storage withinan acceptable temperature range. Potential carriers include, but are notlimited to, physiologically balanced culture medium, phosphate buffersaline solution, water, emulsions (e.g., oil/water or water/oilemulsions), various types of wetting agents, cryoprotective additives orstabilizers such as proteins, peptides or hydrolysates (e.g., albumin,gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g.,sodium glutamate), or other protective agents. The resulting aqueoussolutions may be packaged for use as is or lyophilized. Lyophilizedpreparations are combined with a sterile solution prior toadministration for either single or multiple dosing.

Formulated compositions, especially liquid formulations, may contain abacteriostat to prevent or minimize degradation during storage,including but not limited to effective concentrations (usually 1% w/v)of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben,and/or propylparaben. A bacteriostat may be contraindicated for somepatients; therefore, a lyophilized formulation may be reconstituted in asolution either containing or not containing such a component.

The pharmaceutical compositions of the disclosure can contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate.

The pharmaceutical composition may optionally include an adjuvant toenhance the immune response of the host. Suitable adjuvants are, forexample, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-Aand derivatives or variants thereof, oil-emulsions, saponins, neutralliposomes, liposomes containing the vaccine and cytokines, non-ionicblock copolymers, and chemokines. Non-ionic block polymers containingpolyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POEblock copolymers, MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), amongmany other suitable adjuvants well known in the art, may be used as anadjuvant (Newman et al., 1998, Critical Reviews in Therapeutic DrugCarrier Systems 15:89-142). These adjuvants have the advantage in thatthey help to stimulate the immune system in a non-specific way, thusenhancing the immune response to a pharmaceutical product.

In some embodiments, the composition can include a recombinantparamyxovirus encoding an RSV F ectodomain from one particular RSVsubgroup or strain and also a recombinant paramyxovirus encoding an RSVF ectodomain from a different RSV subgroup or strain. For example, thecomposition can include recombinant paramyxovirus including recombinantRSV F proteins from subtype A and subtype B RSV. The different vectorscan be in an admixture and administered simultaneously, or administeredseparately. Due to the phenomenon of cross-protection among certainstrains of RSV, immunization with one paramyxovirus encoding a RSV Fectodomain from a first strain may protect against several differentstrains of the same or different subgroup.

In some instances it may be desirable to combine a recombinant viralvector, or a composition thereof, with other pharmaceutical products(e.g., vaccines) which induce protective responses to other agents,particularly those causing other childhood illnesses. For example, acomposition including a recombinant paramyxovirus as described hereincan be can be administered simultaneously (typically separately) orsequentially with other vaccines recommended by the Advisory Committeeon Immunization Practices (ACIP; cdc.gov/vaccines/acip/index.html) forthe targeted age group (e.g., infants from approximately one to sixmonths of age). These additional vaccines include, but are not limitedto, IN-administered vaccines. As such, a recombinant paramyxovirusincluding a recombinant RSV F protein described herein may beadministered simultaneously or sequentially with vaccines against, forexample, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP),pneumococcal bacteria (PCV), Haemophilus influenzae type b (Hib), polio,influenza and rotavirus.

Recombinant paramyxoviruses for use in an immunogenic composition, suchas for example a vaccine, are selected based on their attenuation andimmunogenicity. These vaccine selection criteria are determinedaccording to well-known methods. Preferably, candidate viruses have astable attenuation phenotype, exhibit replication in an immunized host,and effectively elicit production of an immune response in a recipient,preferably a protective immune response. Preferably, the candidateviruses stimulate and expand the immune response, e.g., induce an immuneresponse against different viral strains or subgroups and/or stimulatean immune response mediated by a different immunologic basis (e.g.,secretory versus serum immunoglobulins, cellular immunity, and thelike).

The pharmaceutical composition typically contains a effective amount ofa disclosed paramyxovirus and can be prepared by conventionaltechniques. Typically, the amount of recombinant virus in each dose ofthe immunogenic composition is selected as an amount which induces animmune response without significant, adverse side effects. In someembodiments, the composition can be provided in unit dosage form for useto induce an immune response in a subject, for example, to prevent PIVand/or RSV infection in the subject. A unit dosage form contains asuitable single preselected dosage for administration to a subject, orsuitable marked or measured multiples of two or more preselected unitdosages, and/or a metering mechanism for administering the unit dose ormultiples thereof. In other embodiments, the composition furtherincludes an adjuvant.

V. Methods of Eliciting an Immune Response

Provided herein are methods of eliciting an immune response in a subjectby administering one or more of the disclosed recombinantparamyxoviruses to the subject. In a particular example, the subject isa human. The immune response can be a protective immune response, forexample a response that prevents or reduces subsequent infection withthe paramyxovirus or the virus of the heterologous gene included in therecombinant paramyxovirus. Elicitation of the immune response can alsobe used to treat or inhibit viral infection and illnesses associatedtherewith. In several embodiments, the method includes administration ofan immunogenic composition including an attenuated recombinantparainfluenza virus including a viral genome including a heterologousgene encoding a recombinant RSV F ectodomain linked to a PIV F proteintransmembrane (TM) domain and cytoplasmic tail.

A subject can be selected for treatment that has, or is at risk fordeveloping a paramyxovirus infection, such as a RSV and/or a PIVinfection, for example because of exposure or the possibility ofexposure to RSV and/or PIV. Following administration of a disclosedimmunogen, the subject can be monitored for paramyxovirus infection orsymptoms associated therewith, or both.

Methods of intra-nasal administration of recombinant paramyxovirus to asubject are known to the person of ordinary skill in the art, as aremethods of selecting subjects for administration, preparing immunogeniccompositions including the recombinant paramyxovirus for intranasaladministration, and evaluating the subject for an immune response to therecombinant paramyxovirus. Exemplary description of such methods can befound, for example, in Karron et al, 2012. Vaccine, 30(26), 3975-3981,which is incorporated by reference herein in its entirety.

Typical subjects intended for treatment with therapeutics and methods ofthe present disclosure include humans, as well as non-human primates andother animals. Because nearly all humans are infected with RSV and PIVby the age of 5, the entire birth cohort is included as a relevantpopulation for immunization. This could be done, for example, bybeginning an immunization regimen anytime from birth to 6 months of age,from 6 months of age to 5 years of age, in pregnant women (or women ofchild-bearing age) to protect their infants by passive transfer ofantibody, family members of newborn infants or those still in utero, andsubjects greater than 50 years of age. The scope of this disclosure ismeant to include maternal immunization. In several embodiments, thesubject is a human subject that is seronegative for RSV or PIV3 specificantibodies. In additional embodiments, the subject is no more than oneyear old, such as no more than 6 months old, no more than 3 months, orno more than 1 month old.

Subjects at greatest risk of RSV and/or PIV infection with severesymptoms (e.g. requiring hospitalization) include children withprematurity, bronchopulmonary dysplasia, and congenital heart diseaseare most susceptible to severe disease. During childhood and adulthood,disease is milder but can be associated with lower airway disease and iscommonly complicated by sinusitis. Disease severity increases in theinstitutionalized elderly (e.g., humans over 65 years old). Severedisease also occurs in persons with severe combined immunodeficiencydisease or following bone marrow or lung transplantation. Thus, thesesubjects can be selected for administration of a disclosed recombinantparamyxovirus.

To identify subjects for prophylaxis or treatment according to themethods of the disclosure, accepted screening methods are employed todetermine risk factors associated with a targeted or suspected diseaseor condition, or to determine the status of an existing disease orcondition in a subject. These screening methods include, for example,conventional work-ups to determine environmental, familial,occupational, and other such risk factors that may be associated withthe targeted or suspected disease or condition, as well as diagnosticmethods, such as various ELISA and other immunoassay methods, which areavailable and well known in the art to detect and/or characterizeparamyxovirus infection. These and other routine methods allow theclinician to select patients in need of therapy using the methods andpharmaceutical compositions of the disclosure. In accordance with thesemethods and principles, a composition can be administered according tothe teachings herein, or other conventional methods known to the personof ordinary skill in the art, as an independent prophylaxis or treatmentprogram, or as a follow-up, adjunct or coordinate treatment regimen toother treatments.

The administration of a disclosed recombinant paramyxovirus can be forprophylactic or therapeutic purpose. When provided prophylactically, theimmunogen can be provided in advance of any symptom, for example inadvance of infection. The prophylactic administration serves to elicitan immune response that can prevent or ameliorate any subsequentinfection. In some embodiments, the methods can involve selecting asubject at risk for contracting a paramyxovirus infection, andadministering an effective amount of a disclosed recombinantparamyxovirus to the subject. The recombinant paramyxovirus can beprovided prior to the anticipated exposure to paramyxovirus so as toelicit an immune response that can attenuate the anticipated severity,duration or extent of an infection and/or associated disease symptoms,after exposure or suspected exposure to the virus, or after the actualinitiation of an infection. In some examples, treatment using themethods disclosed herein prolongs the time of survival of the subject.

Administration of the disclosed recombinant paramyxoviruses includingRSV and PIV antigens to a subject can elicit the production of an immuneresponse that is protective against serious lower respiratory tractdisease, such as pneumonia and bronchiolitis, or croup, when the subjectis subsequently infected or re-infected with a wild-type RSV or PIV.While the naturally circulating virus is still capable of causinginfection, particularly in the upper respiratory tract, there is areduced possibility of rhinitis as a result of the vaccination and apossible boosting of resistance by subsequent infection by wild-typevirus. Following vaccination, there are detectable levels of hostengendered serum and secretory antibodies which are capable ofneutralizing homologous (of the same subgroup) wild-type virus in vitroand in vivo. In many instances the host antibodies will also neutralizewild-type virus of a different, non-vaccine subgroup. To achieve higherlevels of cross-protection, for example, against heterologous strains ofanother subgroup, subjects can be vaccinated with a compositionincluding recombinant viral vectors including RSV F proteins from atleast one predominant strain of both RSV subgroups A and B.

The recombinant viral vectors described herein, and immunogeniccompositions thereof, are provided to a subject in an amount effectiveto induce or enhance an immune response against the antigens included inthe virus in the subject, preferably a human. An effective amount willallow some growth and proliferation of the virus, in order to producethe desired immune response, but will not produce viral-associatedsymptoms or illnesses. Based on the guidance provided herein andknowledge in the art, persons skilled in the art will readily be able todetermine the proper amount of virus to use in the live vaccine. Theprecise amounts will depend on several factors, for example, thesubject's state of health and weight, the mode of administration, thedegree of attenuation of the virus, the nature of the formulation, andwhether the immune system of the subject is compromised.

An immunogenic composition including one or more of the disclosedrecombinant paramyxoviruses can be used in coordinate (or prime-boost)vaccination protocols or combinatorial formulations. In certainembodiments, novel combinatorial immunogenic compositions and coordinateimmunization protocols employ separate immunogens or formulations, eachdirected toward eliciting an anti-viral immune response, such as animmune response to RSV and PIV proteins. Separate immunogeniccompositions that elicit the anti-viral immune response can be combinedin a polyvalent immunogenic composition administered to a subject in asingle immunization step, or they can be administered separately (inmonovalent immunogenic compositions) in a coordinate (or prime-boost)immunization protocol.

It is contemplated that there can be several boosts, and that each boostcan be a different disclosed immunogen. It is also contemplated in someexamples that the boost may be the same immunogen as another boost, orthe prime.

Upon administration of a disclosed recombinant paramyxovirus the immunesystem of the subject typically responds to the immunogenic compositionby producing antibodies specific for viral protein. Such a responsesignifies that an immunologically effective dose was delivered to thesubject.

For each particular subject, specific dosage regimens can be evaluatedand adjusted over time according to the individual need and professionaljudgment of the person administering or supervising the administrationof the immunogenic composition. In some embodiments, the antibodyresponse of a subject will be determined in the context of evaluatingeffective dosages/immunization protocols. In most instances it will besufficient to assess the antibody titer in serum or plasma obtained fromthe subject. Decisions as to whether to administer booster inoculationsand/or to change the amount of therapeutic agent administered to theindividual can be at least partially based on the antibody titer level.The antibody titer level can be based on, for example, an immunobindingassay which measures the concentration of antibodies in the serum whichbind to an antigen including, for example, an RSV F protein. The actualdosage of disclosed immunogen will vary according to factors such as thedisease indication and particular status of the subject (for example,the subject's age, size, fitness, extent of symptoms, susceptibilityfactors, and the like), time and route of administration, other drugs ortreatments being administered concurrently, as well as the specificpharmacology of the composition for eliciting the desired activity orbiological response in the subject. Dosage regimens can be adjusted toprovide an optimum prophylactic or therapeutic response.

Determination of effective dosages is typically based on animal modelstudies followed up by human clinical trials and is guided byadministration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject, orthat induce a desired response in the subject (such as a neutralizingimmune response). Suitable models in this regard include, for example,murine, rat, porcine, feline, ferret, non-human primate, and otheraccepted animal model subjects known in the art. Alternatively,effective dosages can be determined using in vitro models (for example,immunologic and histopathologic assays). Using such models, onlyordinary calculations and adjustments are required to determine anappropriate concentration and dose to administer a therapeuticallyeffective amount of the composition (for example, amounts that areeffective to elicit a desired immune response or alleviate one or moresymptoms of a targeted disease). In alternative embodiments, aneffective amount or effective dose of the composition may simply inhibitor enhance one or more selected biological activities correlated with adisease or condition, as set forth herein, for either therapeutic ordiagnostic purposes. In one embodiment, a general range of virusadministration is about 10³ to about 10 plaque forming units (PFU) ormore of virus per human subject, including about 10⁴ to about 10⁵ PFUvirus per human subject.

Administration of an immunogenic composition that induces an immuneresponse to reduce or prevent an infection, can, but does notnecessarily completely, eliminate such an infection, so long as theinfection is measurably diminished, for example, by at least about 50%,such as by at least about 70%, or about 80%, or even by about 90% theinfection in the absence of the agent, or in comparison to a referenceagent. Those in need of treatment include the general population and/orpatients infected with or at risk of infection with a paramyxovirus,such as RSV and/or PIV

In one example, a desired response is to inhibit or reduce or preventRSV and/or PIV infection or reinfection. The RSV and/or PIV infectiondoes not need to be completely eliminated or reduced or prevented forthe method to be effective. For example, administration of an effectiveamount of a disclosed recombinant paramyxovirus can decrease subsequenceRSV and/or PIV infection (for example, as measured by infection ofcells, or by number or percentage of subjects infected by RSV and/orPIV) by a desired amount, for example by at least 10%, at least 20%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100% (elimination orprevention of detectable RSV and/or PIV infection, as compared to asuitable control.

The dosage and number of doses will depend on the setting, for example,in an adult or any one primed by prior paramyxovirus infection orimmunization, a single dose may be a sufficient booster. In naivesubjects, in some examples, at least two doses can be given, forexample, at least three doses. In some embodiments, an annual boost isgiven, for example, along with an annual influenza vaccination.

Following immunization of a subject, serum can be collected from thesubject at appropriate time points, frozen, and stored for assay ofantibody titer and/or neutralization testing. Quantification of antibodylevels can be performed by subtype-specific Neutralization assay orELISA. Methods to assay for neutralization activity are known to theperson of ordinary skill in the art and are further described herein,and include, but are not limited to, plaque reduction neutralization(PRNT) assays, microneutralization assays, flow cytometry based assays,single-cycle infection assays. In some embodiments, the serumneutralization activity can be assayed using a panel of RSV or PIVpseudoviruses. Virus-neutralizing antibody titres were determined inserum samples by a PRVN assay as described previously (de Graaf et al.,J. Virol Methods, 143: 169-174, 2007). In brief, serum samples can bediluted and incubated for 60 min at 37° C. with approximately 50 p.f.u.of NL/1/00 or NL/1/99, expressing an enhanced green fluorescent protein.Subsequently, the virus-serum mixtures are added to Vero-118 cells in24-well plates and incubated at 37° C. After 2 h, the supernatants arereplaced by a mixture of equal amounts of infection medium and 2% methylcellulose. Six days later, fluorescent plaques are counted using aTyphoon 9410 Variable Mode Imager (GE Healthcare). Antibody titres areexpressed as the dilution resulting in 50% reduction of the number ofplaques, calculated according to the method of Reed & Muench, Am. J.Hyg., 27, 493-497, 1938.

Additional Embodiments

Clause 1. A recombinant paramyxovirus, comprising (a) a viral genomecomprising a heterologous gene encoding the ectodomain of a type Itransmembrane protein of a heterologous virus linked to thetransmembrane domain (TM) and cytoplasmic tail (CT) of the F protein ofthe paramyxovirus; or (b) a viral genome comprising a heterologous geneencoding the ectodomain of a type II transmembrane protein of aheterologous virus linked to the TM and CT of the HN protein of theparamyxovirus.

Clause 2. The recombinant paramyxovirus of clause 1, wherein therecombinant paramyxovirus is a recombinant human/bovine parainfluenzavirus 3 (B/HPIV3), a recombinant human parainfluenza virus 1 (HPIV1), arecombinant human parainfluenza virus 1 (HPIV2), a recombinant humanparainfluenza virus 1 (HPIV3), a recombinant parainfluenza virus 5(PIV5) a recombinant Sendai virus, or a recombinant Newcastle diseasevirus (NDV).

Clause 3. The recombinant paramyxovirus of clause 2, comprising: arecombinant parainfluenza virus (PIV) comprising a viral genomecomprising a heterologous gene encoding a recombinant respiratorysyncytial virus (RSV) F ectodomain linked to a PIV F protein TM and CT;a recombinant NDV comprising a viral genome comprising a heterologousgene encoding a recombinant RSV F ectodomain linked to a NDV F proteinTM and CT; or a recombinant Sendai virus comprising a viral genomecomprising a heterologous gene encoding a recombinant RSV F ectodomainlinked to a Sendai virus F protein TM and CT.

Clause 4. The recombinant paramyxovirus of any of clauses 1-3,comprising: a recombinant PIV comprising a viral genome comprising aheterologous gene encoding a recombinant RSV F ectodomain linked to aPIV F protein TM and CT.

Clause 5. The recombinant paramyxovirus of clause 4, wherein the RSV Fectodomain is from a human RSV (hRSV) F protein.

Clause 6. The recombinant paramyxovirus of clause 4 or clam 5, whereinthe hRSV F protein is from a subtype A hRSV or subtype B hRSV.

Clause 7. The recombinant paramyxovirus of any one of clauses 4-6,wherein the RSV F ectodomain is stabilized in a RSV Fprefusion-conformation by one or more amino acid substitutions comparedto a native RSV F protein sequence.

Clause 8. The recombinant paramyxovirus of any one of clauses 4-7,wherein the RSV F ectodomain comprises amino acids set forth as: (a)66E; (b) 101P; (c) 155C and 290C; (d) 190F; (e) 207L; or (f) acombination of (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a),(d), and (e); (a), (c), (d), and (e); (a), (b), and (c); (a), (b), and(d); (a), (b), and (e); (a), (b), (e), and (d); (a), (b), (c), (d), and(e); (c) and (d); or (c) and (e); or (c), (d), and (e), wherein theamino acid numbering corresponds to the RSV F protein sequence set forthas SEQ ID NO: 1.

Clause 9. The recombinant paramyxovirus of clause 8, wherein the RSV Fectodomain comprises amino acid substitutions are set forth as: (a)K66E; (b) Q101P; (c) S155C and S290C; (d) S190F; (e) V207L; or (f) acombination of (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a),(d), and (e); (a), (c), (d), and (e); (a), (b), and (c); (a), (b), and(d); (a), (b), and (e); (a), (b), (e), and (d); (a), (b), (c), (d), and(e); (c) and (d); or (c) and (e); or (c), (d), and (e).

Clause 10. The recombinant paramyxovirus of clause 8 or clause 9,wherein the RSV F ectodomain comprises 66E, 101P, 115C, 290C, 190F, and207L.

Clause 11. The recombinant paramyxovirus of any one of clauses 4-10,wherein the RSV F ectodomain comprises an amino acid sequence at least85% identical to the RSV ectodomain of one of SEQ ID NOs: 1 (WT RSV FA), 2 (WT RSV F B), 12 (A2 HEK), 14 (A2 HEK+DS), or 21 (A2 HEK+DS-Cav1),or comprises the amino acid sequence of the RSV ectodomain of SEQ ID NO:12, 14, or 21.

Clause 12. The recombinant paramyxovirus of any one of clauses 4-11,wherein the PIV is a recombinant PIV1, a recombinant PIV2, or arecombinant PIV3.

Clause 13. The recombinant paramyxovirus of clause 12, wherein therecombinant PIV is: a recombinant PIV1, and the TM and CT linked to theRSV F ectodomain are from a PIV1 F protein; a recombinant PIV2, and theTM and CT linked to the RSV F ectodomain are from a PIV2 F protein; or arecombinant PIV3, and the TM and CT linked to the RSV F ectodomain arefrom a PIV3 F protein.

Clause 14. The recombinant paramyxovirus of clause 12 or clause 13,wherein the recombinant PIV is: a recombinant HPIV1 and the PIV F TM andCT linked to the RSV F ectodomain are from a HPIV1 F protein; arecombinant HPIV2 and the PIV F TM and CT linked to the RSV F ectodomainare from a HPIV2 F protein; a recombinant HPIV3 and the PIV F TM and CTlinked to the RSV F ectodomain are from a HPIV3 F protein; or arecombinant B/HPIV3 and the PIV F TM and CT linked to the RSV Fectodomain are from a BPIV3 F protein.

Clause 15. The recombinant paramyxovirus of any one of clauses 4-14,wherein the RSV F ectodomain is from a hRSV F protein, and the TM and CTare from a BPIV3 F protein.

Clause 16. The recombinant paramyxovirus of any one of clauses 4-15,wherein the recombinant PIV is: a recombinant HPIV1 and the PIV F TM andCT linked to the RSV F ectodomain comprise the amino acid sequence setforth as SEQ ID NO: 31, or an amino acid sequence at least 90% identicalto SEQ ID NO: 31; a recombinant HPIV2 and the PIV F TM and CT linked tothe RSV F ectodomain comprise the amino acid sequence set forth as SEQID NO: 39, or an amino acid sequence at least 90% identical to SEQ IDNO: 39; a recombinant HPIV3 and the PIV F TM and CT linked to the RSV Fectodomain comprise the amino acid sequence set forth as SEQ ID NO: 46,or an amino acid sequence at least 90% identical to SEQ ID NO: 46; or arecombinant B/HPIV3 and the PIV F TM and CT linked to the RSV Fectodomain comprise the amino acid sequence set forth as SEQ ID NO: 53,or an amino acid sequence at least 90% identical to SEQ ID NO: 53.

Clause 17. The recombinant paramyxovirus of any one of clauses 4-16,wherein the recombinant PIV is: a recombinant HPIV3 and the heterologousgene encodes a hRSV F ectodomain linked to a HPIV3 F TM and CTcomprising the amino acid sequence set forth as SEQ ID NO: 10, or anamino acid sequence at least 90% identical thereto; or a recombinantB/HPIV3 and the heterologous gene encodes a hRSV F ectodomain linked toa BPIV3 F TM and CT comprising the amino acid sequence set forth as SEQID NO: 21, or an amino acid sequence at least 90% identical thereto.

Clause 18. The recombinant paramyxovirus of any one of clauses 4-17,wherein the RSV F ectodomain is from a hRSV F protein and therecombinant PIV comprises a viral genome encoding: HPIV3 F and HNproteins and BPIV3 N, P, C, V, M, and L proteins, and wherein the TM andCT linked to the RSV F ectodomain are from a BPIV3 F protein; HPIV1 N,P, C, M, F, HN and L proteins, and wherein the TM and CT linked to theRSV F ectodomain are from a HPIV1 F protein; HPIV2 N, P, V, M, F, HN andL proteins, and wherein the TM and CT linked to the RSV F ectodomain arefrom a HPIV2 F protein; or HPIV3 N, P, C, M, F, HN and L proteins, andwherein the TM and CT linked to the RSV F ectodomain are from a HPIV3 Fprotein.

Clause 19. The recombinant paramyxovirus of any one of clauses 4-18,wherein the recombinant RSV F ectodomain linked to the PIV TM and CT isencoded by the first or second gene downstream of a genomic promoter ofthe PIV genome.

Clause 20. The recombinant paramyxovirus of clause 18 or clause 19,wherein the viral genome comprises, from upstream to downstream: a PIVgenomic promoter followed by the N, P, C/V, M, F, HN, and L genes; andwherein the gene encoding the recombinant RSV F ectodomain linked to thePIV TM and CT is located between the genomic promoter and the geneencoding the N protein, or between the genes encoding the N and the Pprotein.

Clause 21. The recombinant paramyxovirus of any one of clauses 18-19,comprising a viral genome encoding: HPIV3 F and HN genes and BPIV3 N, P,C, V, M, and L genes comprising the amino acid sequences set forth asSEQ ID NOs: 21, 101, 47, 48, 49, 52, respectively, or sequences at least90% identical thereto.

Clause 22. The recombinant paramyxovirus of any one of the priorclauses, wherein the heterologous gene is codon-optimized for expressionin human cells.

Clause 23. The recombinant paramyxovirus of clause 22, wherein therecombinant paramyxovirus is: a recombinant HPIV3 and the heterologousgene encodes an RSV F ectodomain linked to a HPIV3 F TM and CT, andcomprises the nucleotide sequence set forth as SEQ ID NO: 11 (GenScriptRSV F_HEK_DS-Cav1_H3TMCT); or a recombinant B/HPIV3 and the heterologousgene encodes an RSV F ectodomain linked to a BPIV3 F TM and CT, andcomprises the nucleotide sequence set forth as SEQ ID NO: 22 (GenArt RSVF_HEK_DS-Cav1_B3TMCT) or SEQ ID NO: 23 (GenScript RSVF_HEK_DS-Cav1_B3TMCT).

Clause 24. A recombinant viral vector, comprising: a viral genomecomprising a heterologous gene encoding a RSV F ectodomain linked to theTM and CT of a type I membrane protein of the viral genome.

Clause 25. The viral vector of clause 24, wherein the RSV F ectodomaincomprises K66E and Q101P amino acid substitutions.

Clause 26. A recombinant viral vector, comprising a viral genomecomprising a heterologous gene encoding a RSV F ectodomain comprisingK66E and Q101P amino acid substitutions.

Clause 27. The viral vector of any one of clauses 24-26, wherein the RSVF protein is stabilized in a prefusion or a postfusion conformation byone or more amino acid substitutions.

Clause 28. The viral vector of any one of clauses 24-27, wherein the RSVF ectodomain is stabilized in the prefusion conformation by S155C,S290C, S190F, and V207L amino acid substitutions

Clause 29. The viral vector of any one of clauses 26-28, wherein the RSVF ectodomain is soluble and can be secreted from a host cell comprisingthe viral vector.

Clause 30. The viral vector of any one of clauses 24-29, wherein theviral vector is a recombinant human/bovine parainfluenza virus 3(B/HPIV3), a recombinant human parainfluenza virus 1 (HPIV1), arecombinant human parainfluenza virus 1 (HPIV2), a recombinant humanparainfluenza virus 1 (HPIV3), a recombinant parainfluenza virus 5(PIV5) a recombinant Sendai virus, or a recombinant Newcastle diseasevirus (NDV).

Clause 31. The viral vector of any one of clauses 24-30, wherein the RSVF ectodomain is from a human RSV (hRSV) F protein.

Clause 32. The viral vector of any one of clauses 24-31, wherein theheterologous gene encoding the RSV F protein comprises the nucleic acidsequence set forth as nucleotides 1-1587 of SEQ ID NO: 18. (ectodomainencoded by GenScript optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA sequence)

Clause 33. The recombinant paramyxovirus or viral vector of any one ofthe prior clauses, wherein at least 90% of viral particles produced by ahost cell infected with the recombinant paramyxovirus or viral vectorcomprise a viral envelope comprising the ectodomain encoded by theheterologous gene.

Clause 34. The recombinant paramyxovirus or viral vector of any one ofthe previous clauses, wherein the recombinant paramyxovirus or viralvector is attenuated.

Clause 35. An immunogenic composition comprising the recombinantparamyxovirus or viral vector of any one of the prior clauses and apharmaceutically acceptable carrier.

Clause 36. The immunogenic composition of clause 35, further comprisingan adjuvant.

Clause 37. A method of eliciting an immune response to a virus and aheterologous antigen encoded thereby in a subject comprisingadministering a therapeutically effective amount of the immunogeniccomposition of clause 35 or clause 36 to the subject.

Clause 38. A method of eliciting an immune response to a paramyxovirusand a heterologous antigen encoded thereby in a subject comprisingadministering a therapeutically effective amount of the immunogeniccomposition of clause 35 or clause 36 to the subject, wherein theimmunogenic composition comprises a recombinant paramyxovirus comprisinga heterologous gene encoding the heterologous antigen.

Clause 39. A method of eliciting an immune response to RSV and PIV in asubject, comprising administering an immunogenic composition comprisinga therapeutically effective amount of the immunogenic composition ofclause 35 or clause 36 to the subject, wherein the immunogeniccomposition comprises a recombinant paramyxovirus comprising aheterologous gene encoding an RSV antigen.

Clause 40. The method of any one of clauses 37-39, wherein the immuneresponse is a protective immune response.

Clause 41. The method of any one of clauses 37-40, comprising aprime-boost administration of the immunogenic composition.

Clause 42. The method of any one of clauses 37-41, comprising intranasalor parenteral administration of the immunogenic composition.

Clause 43. The method of any one of clauses 37-42, wherein the subjectis a human or a veterinary subject.

Clause 44. The method of any one of clauses 37-43, wherein the subjectis at risk of or has a RSV or a PIV infection.

Clause 45. The method of any one of clauses 37-44, wherein the subjectis less than one year old.

Clause 46. A nucleic acid molecule comprising the genome of therecombinant paramyxovirus of any one of clauses 1-25.

Clause 47. A recombinant RSV F protein or immunogenic fragment thereofcomprising K66E and Q101P amino acid substitutions.

Clause 48. The recombinant RSV F protein or immunogenic fragment thereofof clause 47, further comprising: (a) S155C and S290C ; (b) S190F; (c)V207L; or (f) a combination of (a) and (b); (a) and (c); (b) and (c); or(a), (b), and (c).

Clause 49. The immunogenic fragment of the recombinant RSV F protein ofclause 47 or clause 48, comprising the RSV F ectodomain.

Clause 50. A nucleic acid molecule encoding the recombinant RSV Fprotein of any one of clauses 47-49.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Improved Expression and Immunogenicity of the RespiratorySyncytial Virus (RSV) Fusion (F) Glycoprotein Expressed by an AttenuatedParainfluenza Virus Vector

This example describes approaches to enhance the immunogenicity andstability of RSV F expressed by a recombinant B/HPIV3 by using RSV Fsequence from an early passage virus, by codon-optimization, by usingstable and highly immunogenic pre-fusion and post-fusion forms of RSV F,and by engineering the RSV F protein TM and CT so that it was moreefficiently incorporated into vector particles.

Introduction.

Live attenuated RSV strains administered represent one strategy for anRSV vaccine, and these are currently under development (Hurwitz. 2011.Expert. Rev. Vaccines. 10:1415-1433; Collins and Melero. 2011. VirusRes. 162:80-99; Karron, et al. 2013. Current Topics Microbiology andImmunology 372:259-284). A live attenuated RSV strain typically would beadministered by the intranasal (IN) route. However attenuation generallyresults in reduced antigen synthesis, resulting in reducedimmunogenicity. Obtaining a suitable balance between attenuation andimmunogenicity has been challenging for RSV.

Complete, infectious HPIVs can be generated entirely from cloned cDNAsin transfected cell culture (using reverse genetics). A foreign genedesigned for expression would be modified so that it is flanked by HPIVtranscription signals (called the gene-start and gene-end signals,located at the beginning and end of each gene, respectively) and wouldbe inserted as an additional gene into the HPIV genome by reversegenetics. The foreign gene would then be transcribed into a separatemRNA, like the other HPIV genes. HPIVs can accommodate and expressseveral added foreign genes (Skiadopoulos, et al. 2002. Virology297:136-152). However, multiple genes can be overly attenuating and cancollect point mutations (Skiadopoulos, et al. 2002. Virology297:136-152).

HPIV transcription initiates at a single promoter at the 3′ end of thegenome and proceeds sequentially. A fraction of the polymerasedisengages from the template at each gene junction, resulting in anegative gradient of gene transcription. Therefore, promoter-proximalgenes are expressed more frequently than downstream genes. Placement ofa foreign gene close to the promoter would increase expression, but hasthe potential to affect expression of downstream vector genes. Otherfeatures, such as differences in the efficiency of gene-start orgene-end transcription signals or effects of other structural featuresin the RNA template that sometimes are present but are poorlyunderstood, also can unpredictably affect expression of an inserted geneor open reading frame (ORF) (Whelan, et al. 2004. Current TopicsMicrobiology and Immunology 283:61-119). In addition, in some cases theproperties of viral constructs can be greatly affected by factors thatremain unidentified; for example, the insertion of the RSV F gene intothe P-M gene junction of a PIV3 vector resulted in a virus that wassubstantially temperature-sensitive and attenuated (Liang B, et al.2014. J Virol 88:4237-4250). Thus, while the broad details of expressionfrom HPIV genomes is generally known, specific constructions can giveunpredictable results.

In previous studies, the B/HPIV3 vector was used as a vector to expressthe RSV G gene and F proteins from added genes in the first and secondgenome positions after the promoter or to express the RSV F gene from anadded gene in the second genome position between the N and P genes. Thelatter virus, called MEDI-534, has been evaluated in clinical studies inseronegative children and was attenuated, well tolerated, and infectiousbut was less immunogenic against RSV than hoped (Bernstein, et al. 2012.Pediatric Infectious Disease Journal 31:109-114). Analysis of shedvaccine virus from vaccine recipients showed that ˜50% of specimenscontained vaccine virus with mutations that would be predicted toperturb RSV F expression. This likely reduced immunogenicity.Retrospective analysis of the clinical trial material (CTM) showed that2.5% of this virus did not express RSV F (Yang, et al. 2013. Vaccine31:2822-2827). In addition, the observation that the RSV F insertaccumulated mutations that inactivated its expression at the proteinlevel, and that these mutations were amplified during growth, suggeststhat there was a selective advantage to silencing expression of the RSVF protein. This likely could be due to the highly fusogenic nature ofthe RSV F protein, which efficiently mediates syncytium formation. Invitro, this results in destruction of the cell substrate, which couldreduce vector replication. In addition, the synthesis of high levels ofa foreign glycoprotein could interfere with the synthesis, processingand transport of the vector glycoproteins through the endoplasmicreticulum and exocytic pathway, and could interfere sterically withvirion morphogenesis, among other things. These effects might occur bothin vitro and in vivo.

Expression of an Early-Passage (HEK) Version of the RSV F Protein andCodon-Optimized Versions of the RSV F Open Reading Frame (ORF).

Increased expression of viral antigen typically provides enhancedimmunogenicity. Codon-optimization of the ORF encoding a vectoredantigen can increase its expression and in turn enhance itsimmunogenicity, for example as has been shown with humanimmunodeficiency virus antigens expressed from viral or DNA vectors(Gao, et al. 2003. AIDS research and human retroviruses 19:817-823;Carnero, et al. 2009. J Virol 83:584-597). However, these sequencechanges can have effects beyond improving translation, such as effectson mRNA stability and transport, and so the effects of altering thenucleotide sequence of an mRNA can be complex and unpredictable.Therefore, a codon-optimized version of the RSV F sequence was designedusing GeneArt (GA) algorithms and was evaluated to determine whether itconferred protein expression.

When designing this codon-optimized ORF, the amino acid sequence of anearly-passage version of RSV strain A2 from the 1960s was mistakenlyused (Connors, et al. 1995. Virology 208:478-484; Whitehead, et al.1998. J Virol 72:4467-4471). This early-passage (or low-passage) strainfrom the 1960s is called HEK after the human embryonic kidney (HEK) cellculture used in its propagation. The HEK virus differed from current,highly passaged laboratory version of RSV strain A2 by two amino acidassignments (Connors, et al. 1995. Virology 208:478-484; Whitehead, etal. 1998. J Virol 72:4467-4471). The HEK version had assignments 66E and101P whereas the highly passaged laboratory A2 strain had assignments66K and 101Q (hereafter called “non-HEK” assignments) (FIG. 1). However,the occurrence of sequence differences between virus strains or betweenstocks of a given strain is common for RNA viruses given their highmutation rate, and the HEK differences previously had no knownimportance. Further, the presence of the HEK assignments in anattenuated RSV vaccine candidate called RSV NIH ΔM2-2 was associatedwith a small reduction in the efficiency of replication in cell culture.Additionally, the HEK assignment at position 66 was identified to affectsyncytium formation during RSV infection. Thus, it was intended to avoidthe HEK assignments given their association with reduced replication.However, because the version of RSV F containing the HEK assignments wasaccidentally used for the initial codon optimization, a parallelGA-optimized non-HEK version was constructed and the two versions werecompared (FIG. 1). The two versions of RSV F were placed under thecontrol of BPIV3 gene-start and gene-end transcription signals andinserted into the 2^(nd) position of the rB/HPIV3 vector (FIG. 1). Thetranscription signals and insert position were used in all subsequentrB/HPIV3 constructs expressing RSV F so as to provide direct comparisonsthroughout.

Vero cells were infected with the two different vectors (called“HEK/GA-opt” and “non-HEK/GA-opt”), cell lysates were prepared 48 hpost-infection, and the proteins were subjected to gel electrophoresisin the presence of denaturing detergent and under reducing ornon-reducing conditions. The separated proteins were transferred tomembranes by Western blotting and were analyzed using antibodiesspecific to RSV F (FIG. 2). This showed that the presence of the HEKassignments was associated with a small (˜2-fold) but consistentincrease in the expression of RSV F protein (FIG. 2). One non-limitingexplanation for this finding is that the HEK assignments increased Fprotein stability although an effect on protein synthesis is possiblebut seems less likely given that the HEK and non-HEK versions of the FORF were identical except for two codons. In addition, when analyzedunder non-reducing conditions, the presence of the HEK assignments wasassociated with a reduction in the gel mobility of the RSV F trimer(FIG. 2). This suggested that these assignments altered the F proteintrimer structure. More strikingly, expression of the HEK version of RSVF was associated with a drastic reduction in syncytium formationcompared to the non-HEK version (FIG. 3) even though the HEK version wasexpressed at a slightly increased level, as already noted. This assaytakes advantage of the general lack of evident syncytia induced in cellsinfected by the rB/HPIV3 empty vector, whereas the expression of the RSVF protein from the vector results in syncytium formation that isgenerally proportional to the amount of expression of RSV F protein.This provides an assay for the quantity and functionality of RSV Fprotein expressed from a PIV vector. These observations concerning HEKindicated that the HEK assignments were associated with differences insynthesis/stability, structure, and fusogenic activity of RSV F, andthat these effects occurred in the absence of any other RSV proteins andthus were directly relevant to expression from a heterologous vector.

Because the HEK assignments are from a low-passage stock of RSV strainA2 from the 1960s, they are likely to be representative of the originalclinical isolate, whereas the non-HEK assignments had appeared duringextensive passage in vitro over subsequent decades. This suggests thatthe hypo-fusogenic phenotype of the HEK version of F is morerepresentative of the original biological virus. The non-HEK version mayrepresent a hyper-fusogenic variant that was selected for during passagein cell culture. A hyper-fusogenic version of RSV F might be lessfavored in nature because it might destabilize the virus, but might beselected for in a laboratory setting of rapid growth in a cellmonolayer. 226 sequences of RSV F from clinical isolates in the GenBankdatabase were examined and it was found that clinical isolates usuallycontained the HEK assignments. This is consistent with these assignmentsbeing representative of circulating RSV. In any event, the HEKassignments provided a modest increase in F protein expression andprovided a form of RSV F that was hypo-fusogenic. The reduction insyncytium formation is advantageous because it reduces cytopathogenicitythat might otherwise interfere with HPIV vector replication and favorselection of vector in which the RSV F insert was silenced. Therefore,the HEK assignments have the triple advantage of representing a morenative and clinically relevant form of the F protein, providing a modestincrease in protein expression, and reducing selective pressure tosilence the RSV F insert.

The effect of codon-optimization on RSV F expression and immunogenicitywas also evaluated. The HEK-containing and GA-optimized version(HEK/GA-opt) described above was used along with two othercodon-optimized RSV HEK F sequences made by two other differentalgorithms. Evaluation of multiple optimized versions is not a typicalpractice, since it increases the expense and inconvenience and had notbeen shown to be useful. The two other sources were DNA2.0 (D2) andGenScript (GS) algorithms; also included for comparison was the non-HEK,non-codon-optimized version (FIG. 4). Codon optimization resulted insignificantly enhanced RSV F protein synthesis that, surprisingly,differed in magnitude for the different versions. The highest expressionwas observed for the HEK-containing GenScript-optimized F protein(HEK/GS-opt), which was 10-fold (Vero cells) and 16-fold (LLC-MK2 cells)higher than the unmodified RSV F (non-HEK/non-opt) (FIG. 5). The levelsof expression with the more efficient ORFs were so high thatprogressively increasing levels of syncytium formation were evident inassociation with increasing levels of F expression despite the presenceof the HEK assignments (FIG. 6), although it can be presumed thatsyncytium formation would have been even faster and more extensive inthe absence of the HEK assignments.

Codon-pair optimization was also evaluated as a means to increase Fprotein expression, using an algorithm that was previously described(Coleman, et al. 2008. Science 320:1784-1787). Codon-pair optimizationincreases the frequency of codon pairs associated with high expression.However, this did not confer any increase in expression in the case ofRSV F.

Contrary to expectations, the 10- to 16-fold increase in RSV Fexpression and concomitant increase in syncytium formation did not havea significant negative impact on vector replication in cell culture(FIG. 7). It might have been anticipated that high levels of RSV Fexpression and syncytium formation would have interfered with the vectorat any of a number of steps, as already noted, including vectorglycoprotein synthesis, processing, exocytosis, vector particleformation, and cell viability, but this was not the case. This wasparticularly surprising because, as already noted, the accumulation andamplification of mutations that silenced expression of the RSV F gene inMEDI-534 suggested that there was a substantial selective pressureagainst expression of RSV F protein. Compared with the empty vector, allvectors with RSV F insert were moderately attenuated (FIG. 7)—perhapsinvolving a common attenuating effect such as the increase in genomelength and gene number—but replicated with similar kinetics to eachother and grew to high peak titers that were slightly lower than thepeak titer of empty vector (FIG. 7). Modest variance of peak titerslikely represents experimental variability.

In vivo replication, immunogenicity, and protective efficacy of therB/HPIV3 vectors was evaluated in a hamster model. Groups of hamsterswere immunized intranasally with the rB/HPIV3 vectors at a dose of 10⁵tissue-culture-infection-dose-50 units (TCID₅₀) per animal. In addition,wildtype (wt) RSV given at a dose of 10⁶ plaque forming units (pfu) wasincluded as positive control for the induction of RSV-specific immunity.The wt RSV control was included with the caveat that wt RSV was anon-attenuated virus whereas the vectors were attenuated and might berelatively less immunogenic for that reason. Six animals per virus perday were euthanized on days 3 and 5 post-infection, and nasal turbinatesand lungs were collected for virus titration to measure replication invivo. This showed that vectors bearing the RSV F insert were moderatelyattenuated in the nasal turbinates (upper respiratory tract), andsubstantially attenuated in the lungs (lower respiratory tract) ascompared with the empty vector (FIG. 8). Increased attenuation comparedto the empty vector was evident by the lower values for virus shedding.It also was evident by comparison of the day 3 and day 5 titers: for theempty vector, the titers on days 3 and 5 were comparable, whereas forthe vectors bearing RSV F, the day 3 titers were lower than the day 5titers, indicating that these constructs took longer to achieve theirmaximum titers. Surprisingly, among the vectors with RSV F insert, thosewith enhanced RSV F expression were not more attenuated than the onewith less RSV F expression, i.e., non-HEK/non-opt. Thus, the addition ofthe RSV F insert to the rB/HPIV3 vector was attenuating in vivo—perhapsdue to some common feature such as the increase in genome length or genenumber—but this did not appear to be substantially influenced by thelevel of synthesis of the RSV F protein.

The immunogenicity of the vectors was assessed by measuring the serumtiters of RSV-neutralizing antibodies by a 60% plaque reduction assaysupplemented with guinea pig complement, which is a standard assay. Allvectors expressing RSV F induced similarly high titers ofRSV-neutralizing serum antibodies, irrespective of HEK assignments orcodon-optimization (FIG. 9). There was a modest progressive increase inneutralizing titers associated with increasing RSV F expression, but thedifferences were not statistically significant. WT RSV that had beeninfected in parallel as a control induced significantly higher titers ofRSV-neutralizing antibodies than the vectors. However, it is importantto note that the neutralizing antibodies induced by RSV infectionincluded contributions from both the F and G neutralization antigens,whereas the vectors only had F-specific antibodies contributing to theneutralizing titers. In addition, the non-attenuated wt RSV controlreplicated more efficiently than the attenuated vectors, especially inthe lungs (FIG. 8), which would have increased its immunogenicitycompared to that of the vectors.

In order to assess the protective efficacy of these vectors, immunizedhamsters in groups of 6 animals, from the experiment in FIG. 9, werechallenged 30 days post-immunization by intranasal infection with 10⁶pfu of wt RSV per animal. Nasal turbinates and lungs were collected fromeuthanized animals at 3 days post-challenge, and tissue homogenates wereprepared and evaluated by plaque assay to measure the levels ofchallenge RSV replication. Vectors expressing RSV F conferred almostcomplete protection in the lungs and intermediate levels of protectionin the nasal turbinates, while wt RSV conferred almost completeprotection in both anatomical sites (FIG. 10). There was no significantdifference among the vectors expressing RSV F in the protective efficacyagainst RSV challenge. It should be noted that the protection conferredby RSV would include contributions from neutralizing antibodies againstboth the F and G proteins as well as cellular immunity againstpotentially all of the RSV proteins, whereas protection conferred by thevectors would include humoral and cellular immunity against solely the Fprotein. In addition, as noted, the RSV control was a non-attenuated wtvirus that replicated to higher titers than the vectors duringimmunization (FIG. 8), especially in the lungs, which would increase itsimmunogenicity and protective efficacy.

These results showed that the 10- to 16-fold increase in expression ofthe RSV F protein expression resulting from the use of the HEKassignments and codon-optimized sequence did not result in a significantincrease in the induction of RSV-neutralizing serum antibodies (althougha trend towards an increase was observed) or a significant increase inprotection against wt RSV challenge. In contrast, a similar level ofincrease in expression for human immunodeficiency virus antigens hadresulted in enhanced protection with other viral vectors and DNAvaccines in different animal models (Gao, et al. 2003. AIDS research andhuman retroviruses 19:817-823; Carnero, et al. 2009. J Virol83:584-597). Previously, it had also been observed that a 30- to 69-folddifference in the expression of RSV F due to insertion at positions 1 or2 versus 6 in the rB/HPIV3 vector induced significant differences in theprotective efficacy in hamsters (Liang B, et al. 2014. J Virol88:4237-4250). Thus, it is generally thought that an increase in antigensynthesis would confer an increase in immunogenicity. However in somecases this effect might not be of sufficient magnitude to be detectedunambiguously, or it may be that a given in vivo model might not besufficiently sensitive. Thus, the 10- to 16-fold difference in thepresent study might not be sufficient to induce an effect of sufficientmagnitude to be statistically significant in the semi-permissive hamstermodel. The beneficial effect of higher RSV F expression might be moreprominent in combination with other features, or in a permissive host,i.e. primates and humans, with a larger sample size in a pre-clinicaland clinical evaluation. In particular, the 10- to 16-fold increase in Fprotein expression observed in this study was in Vero (African greenmonkey) or LLC-MK2 (rhesus monkey) cells, in which codon-optimizationfor human use would likely be effective given the relatively closephylogenetic relatedness of these primates to humans. In contrast, thein vivo immunogenicity assay employed hamsters, in which codonoptimization for human use might not be effective in increasingexpression and, thereby, immunogenicity.

Evaluation of the Immunogenicity of the Pre-Fusion and Post-Fusion Formsof RSV F Expressed by the rBfHPIV3 Vector.

Like all paramyxovirus F proteins, the RSV F protein initially assemblesinto a pre-fusion conformation that is the version that initiallyaccumulates on the surface of infected cells and is incorporated intovirions. Pre-fusion F can be triggered, such as by contact with anadjacent target cell membrane, to undergo massive conformational changesthat mediate membrane fusion, with the F protein ending in a post-fusionconformation (Calder, et al. 2000. Virology 271:122-131; McLellan, etal. 2013. Science 340:1113-1117; McLellan, et al. 2011. J Virol85:7788-7796; Swanson, et al. 2011. Proc. Nat'l Acad. Sci. U.S.A.108:9619-9624). The RSV F protein is notable among the paramyxovirusesfor being highly susceptible to triggering and can readily be triggeredprematurely, which may contribute to the marked instability of RSVinfectivity. There also is evidence that much of the RSV F protein thataccumulates in infected cells is conformationally heterogeneous, whichmay act as a decoy to reduce the induction of virus-neutralizingantibodies (Sakurai, et al. 1999. J Virol 73:2956-2962). Therefore, itwould be advantageous for more than one reason to express RSV F in astabilized conformation.

Recently, a stable post-fusion form of RSV F was described (McLellan, etal. 2011. J Virol 85:7788-7796; Swanson, et al. 2011. Proc. Nat'l Acad.Sci. U.S.A. 108:9619-9624). This stable post-fusion form was generatedrecombinantly by truncation of the hydrophobic fusion peptide andremoval of the C-terminal transmembrane domain (TM) and cytoplasmic tail(CT) (Ruiz-Arguello, et al. 2004. J General Virology 85:3677-3687). Withthe lack of the TM and CT, this post-fusion form would not bemembrane-anchored and would be secreted. The post-fusion form of RSV Fhas been shown to be immunogenic and protective in mice (Swanson, et al.2011. Proc. Nat'l Acad. Sci. U.S.A. 108:9619-9624).

However, it is thought that the pre-fusion form of RSV F is much moreimmunogenic than the post-fusion form (McLellan, et al. 2013. Science340:1113-1117). This is based on the observation that the vast majorityof the neutralizing activity in convalescent animal and human sera wasconferred by antibodies that do not bind to the post-fusion F proteinand presumably are specific to the pre-fusion form (McLellan, et al.2013. Science 340:1113-1117; Magro, et al. 2012. Proc Nat'l Acad. Sci.U.S.A. 109:3089-3094). Recently, the structure of the pre-fusion form ofRSV F was determined, and it became possible to stabilize thispre-fusion conformation through structure-based mutations: one of theseinvolves the introduction of a disulfide bond (DS), and another involvesamino acid substitutions in a predicted cavity in the timer structure(Cav1), and the combination of these is called DS-Cav1 (McLellan, et al.2013. Science 342:592-598). The recombinant DS and DS-Cav1 forms of theRSV F protein were evaluated as subunit vaccines in mice and macaquesand were shown to induce significantly higher levels of RSV neutralizingserum antibodies than the post-fusion form, with the DS-Cav1 form beingmore immunogenic than the DS form (McLellan, et al. 2013. Science342:592-598).

The immunogenicity of post-fusion and pre-fusion forms of RSV F whenexpressed from the live attenuated rB/HPIV3 vector was evaluated. Thepost-fusion and stabilized pre-fusion forms (DS and DS-Cav1) of RSV Fwith HEK assignments were GA codon-optimized and inserted into the2^(nd) genome position of the rB/HPIV3 vector (FIG. 11). These werecompared with HEK/GA-opt as well as with a version of HEK-containing,GA-optimized F protein from which the CT and TM had been deleted,leaving the ectodomain (Ecto) (FIG. 11). These constructs were alsocompared to the non-HEK/non-opt construct.

GA-optimized F ORF was used in the data presented in FIG. 11 andsubsequent experiments. Parallel constructs with the GS-optimized ORFhave been constructed in some instances (see FIG. 35) but remain to beevaluated. Given the superior expression of the GS-optimized ORF (FIG.5), GS-optimized versions may be more immunogenic and protective. Also,the identifiers “HEK” and “GA-opt” are sometimes omitted from constructnames in in FIG. 11 and subsequent Figures and in the subsequent textfor the sake of simplicity, but the presence of these features isindicated in the Figures (i.e. “All above versions of RSV F are HEK,GA-optimized”, FIG. 11).

Vectors with these various forms of RSV F were rescued, and each grew tohigh, similar titers in vitro (FIG. 12). These were generally slightlyattenuated in terms of growth kinetics and final yield compared to theempty rB/HPIV3 vector, as was noted previously for other vectorconstructs (see FIG. 7).

The efficiency of expression of the various forms of RSV F protein wasevaluated in Vero and LLC-MK2 cells infected with the various constructs(FIGS. 13A and B). Infected cell cultures were harvested 48 hpost-infection and analyzed by Western blotting. The native form of RSVF (i.e., HEK/GA-opt) was cell-associated, as expected. The post-fusionand Ecto forms were found to be secreted as well as to becell-associated. The secretion of post-fusion F was consistently moreefficient than that of the Ecto form: the latter might remain morecell-associated because it contained a higher content of hydrophobicsequence. Unexpectedly, the DS and DS-Cav1 forms of F were expressedmore efficiently (FIG. 13B). Since these viruses replicated at similarkinetics and since the ORFs were similarly GA-optimzed, this increase inexpression likely reflected increased protein stability of the DS andDS-Cav1 forms. Protein engineering can substantially affect theexpression, processing, and stability of a glycoprotein, and often in anegative way, and so the efficient expression of DS and DS-Cav1 by thislive vector was an essential property that could not have been reliablypredicted.

Replication of these vectors in vivo was evaluated in hamsters (FIG.14). In the nasal turbinates, all vectors with RSV F inserts weremoderately more attenuated than the empty vector (FIG. 14A). Increasedattenuation compared to the empty vector was evident by the lower valuesfor virus shedding. It also was evident by comparison of the day 3 andday 5 titers: for the empty vector, these values were comparable,whereas for the vectors bearing RSV F, the day 5 titers were higher thanthe day 3 titers, indicating that these constructs took longer toachieve their maximum titers. The vector with post-fusion F replicatedto a higher titer than those with other forms of F whereas the vectorwith pre-fusion F (DS) replicated to a lower titer, which mightrepresent experimental variability or might represent authenticdisparate effects on vector replication. In the lungs, all vectors withRSV F insert were substantially more attenuated than the empty vector(FIG. 14B). Consistent with the nasal turbinates, the vector withpost-fusion F also replicated to somewhat higher titer than othervectors in the lungs whereas the vector expressing the pre-fusion (DS)version appeared to be somewhat more attenuated. The RSV control, whichis a fully wt virus, replicated more efficiently than the attenuatedrB/HPIV3 vectors expressing RSV F; for example, wt RSV replicated to100- and 1000-fold higher titers in the nasal turbinates and lungs,respectively, than the vector expressing pre-fusion (DS) F.RSV-neutralizing serum antibody titers were determined by a 60% plaquereduction assay. This was performed in two ways: (i) in the presence ofadded complement (which is the usual practice, as already shown in FIG.9), and (ii) in the absence of added complement (FIGS. 15 A and B,respectively). The presence of added complement provides for the mostsensitive detection of virus-specific antibodies, since complementpotentially confers viral-lysis capability to all antibodies that boundto the virion, and also can exert steric effects (Yoder et al 2004 J MedVirol 72:688-694). In contrast, the complement-independentneutralization assay would detect only high quality neutralizingantibodies that are able to neutralize RSV without involving theviral-lysis function or steric effects of the complement proteins. Ithas been suggested that “neutralization assays performed withoutcomplement may be most reflective of physiologic conditions in therespiratory tract” (Yoder et al 2004 J Med Virol 72:688-694). In thecomplement-containing assay (FIG. 15A), vector with post-fusion F waspoorly immunogenic among the vectors even though it replicated to thehighest titers in hamsters; while the vector with pre-fusion (DS) F wasthe most immunogenic among the tested vectors even though it was themost attenuated. In the complement-independent assay (FIG. 15B), amongthe vectors, only the one expressing pre-fusion (DS) F induced hightiters of neutralizing antibodies. None of the other vectors withunmodified, post-fusion, or Ecto F were effective at inducing highquality neutralizing antibodies. The wt RSV control was efficient atinducing antibodies that were neutralizing in both thecomplement-containing and complement-independent assays. Remarkably, thevector with pre-fusion (DS) F was statistically similar to wt RSV ininducing high-quality RSV neutralizing antibodies (FIG. 15B). This isnoteworthy because this attenuated vector replicated 100 to 1000 timesless efficiently than non-attenuated wt RSV and the neutralizingactivity conferred by wt RSV had the additional contribution from theRSV G protein. This suggested that the vector expressing pre-fusion (DS)form of RSV F was very potent and highly immunogenic in inducing veryeffective neutralizing antibodies.

In order to assess the protective efficacy of these vectors, immunizedhamsters from the experiment in FIG. 15 were challenged 30 dayspost-immunization by intranasal infection with 10⁶ pfu of wt RSV peranimal. The animals were sacrificed 3 days post infection and nasalturbinates and lungs were harvested and processed into tissuehomogenates that were assayed by plaque titration (FIG. 16). In thenasal turbinates (FIG. 16A), constructs expressing non-HEK/non-opt F, orHEK/GA-opt, or Ecto F, conferred a moderate level of protection, whereaspost-fusion F and especially pre-fusion (DS) F were somewhat moreprotective. In the lungs (FIG. 16B), all of the vector constructsprovided substantial protection against RSV challenge, except thepost-fusion form, which conferred the least protection (FIG. 16B). Thewt RSV control provided nearly-complete protection in the nasalturbinates and complete protection in the lungs; however, as alreadynoted, wt RSV had the advantage of expressing both the F and Gneutralization antigens, in addition to expressing all of the RSVproteins as potential antigens for cellular immunity, as well asreplicating up to 1000-fold more efficiently (FIG. 14).

The addition of the Cav-1 mutations to the DS construct providedincreased immunogenicity as a subunit vaccine (McLellan, et al. 2013.Science 342:592-598) and is anticipated to further enhance theimmunogenicity of the pre-fusion RSV F expressed from a viral vector.Also, the DS and DS-Cav1 forms of RSV F remain to be evaluated forimmunogenicity and protective efficacy in the context of theGS-optimization, which provided the greatest increase in expression(FIGS. 5 and 6). These further constructs have been constructed andrecovered and prepared as working pools (FIG. 35).

Enhancing the Immunogenicity of RSV F Protein by Facilitating itsIncorporation into the Virion Particle of the rB/HPIV3 Vector.

The incorporation of antigens into virus like particles (VLP) oradeno-associated virus particles has been shown to increase theirimmunogenicity (Rybniker, et al. 2012. J Virol 86:13800-13804; McGinnes,et al. 2011. J Virol 85:366-377). But whether the incorporation of aheterologous antigen into the viral envelope of an infectious viruscould enhance its immunogenicity was unclear. When expressed byrB/HPIV3, the native RSV F protein (i.e., HEK/GA-opt) is incorporatedinto the vector particle only in trace amounts (see below).

A previous study by Zimmer et al (Zimmer et al J Virol 2005 79:10467-77)evaluated the expression of RSV F protein from an added gene in Sendaivirus, which is a murine relative of HPIV1 and also is closely relatedto HPIV3. That study showed that, as with rB/HPIV3, very little RSV Fprotein was incorporated into the Sendai virus vector particle. Theinvestigators replaced the CT or CT plus TM of the RSV F protein withthe corresponding sequences from the Sendai F protein on the premisethat this would improve the efficiency of interaction of the foreign RSVF protein with the vector particle. These modifications indeed increasedincorporation of the engineered RSV F into the Sendai particle, but onlyif the Sendai F protein gene was deleted. That requirement to delete thevector F protein would be undesirable in the present study becausedeleting the vector F protein from rB/HPIV3 would have the potential ofsubstantially altering its replicative properties, especially in vivo,and also would remove one of the HPIV3 protective antigens.

Despite this clear precedent indicating that this strategy would be notbe suitable, rB/HPIV3 constructs were made in which the RSV F proteinhad CT or CT plus TM replaced with that of the vector(PIV) F protein(resulting in constructs called B3CT and B3TMCT, respectively, FIG. 17).The TM and CT regions from rB/HPIV3 were 21 and 26 amino acids in lengthrespectively. The constructions in the present study were done with theversion of the F protein that contained the HEK assignment and GAoptimization in native F protein (HEK/GA-opt), and also with thepre-fusion DS and DS-Cav1 forms (FIG. 17). (The GA-optimized F ORF wasused). All chimeric F genes were inserted into the 2^(nd) position ofrB/HPIV3 for direct comparison with the constructs described above.

All of the viruses were readily recovered by reverse genetics. Toquantify the packaging efficiency of RSV F and its modified derivatives,sucrose-purified viruses were prepared for Western blot analysis todetermine the amount of RSV F in the particle (FIG. 18). Equal amountsof each sucrose-purified stock (0.5 ug protein per sample) weresubjected to denaturing, reducing gel electrophoresis and analyzed byWestern blotting. This showed that non-chimeric F protein (fromHEK/GA-opt) had relatively poor incorporation into the rB/HPIV3 virions(FIG. 18, lane 2). However, the B3CT and B3TMCT modificationsdramatically enhanced the incorporation efficiency by 19- to 20-fold(FIG. 18, lanes 3 and 4). Indeed, when compared to an equal protein massof wt RSV virions (FIG. 18, lane 5), the amount of incorporated B3CT andB3TMCT F protein in the vector particles appeared to be equal to theamount of native F in the RSV particles. Similarly enhanced efficiencyof packaging also was observed for the chimeric pre-fusion (DS) form ofRSV F with B3CT or B3TMCT (FIG. 18, lanes 6 and 7). Thus, efficientpackaging of RSV F B3CT and B3TMCT into the rB/HPIV3 vector did notrequire deletion of the vector F protein, and thus differed dramaticallyfrom the Sendai precedent.

Packaging of RSV F also was examined with transmission electronmicroscopy (TEM) using RSV-specific antibody and immune-gold labeling(FIG. 19 A-F). RSV F spikes on the surface of RSV particles were labeled(FIG. 19A), while no labeling could be observed on the surface of theempty rB/HPIV3 vector (FIG. 19B). Very limited labeling of native RSV Fwas detected in the vector envelope (FIG. 19C), consistent with theresults in FIG. 18 showing that very little native F was detected inpurified rB/HPIV3 virions by Western blot analysis. In contrast, vectorsexpressing chimeric F with B3CT or B3TMCT showed enhanced labeling(FIGS. 19D and E), indicating efficient packaging of these chimericforms into the vector envelope. Likewise, the pre-fusion (DS) RSV F withB3TMCT also was efficiently packaged into the vector particles (FIG.19F). This confirmed that the B3CT and B3TMCT modifications resulted indramatically increased incorporation of RSV F into the rB/HPIV3particles. In addition, this showed that the incorporated RSV F proteinwas present in immunologically active surface spikes similar inappearance to those of authentic RSV particles. Furthermore, stabilizedpre-fusion DS F protein also efficiently appeared at the virion surface.

The high efficiency of packaging of RSV F B3CT and B3TMCT into thevector particles raised the possibility that this would be attenuatingto vector replication, since it is generally assumed that a virionsurface is organized for efficiency and is limited in its capacity forsurface proteins, so that changes in the composition of surface proteinscould be attenuating, especially since the modified B3CT and B3TMCT RSVF proteins contained the CT or the TMCT regions of vector F protein thatare thought to interact with internal viral proteins. For example,efficient incorporation of RSV F into the vector envelope might displacevector HN and F glycoproteins, or might interfere with interactionsbetween vector components (such as between the vector F and HNglycoproteins and the internal M protein during virion assembly, orbetween the vector F and HN proteins that must interact to efficientlyinitiate virus entry). Surprisingly, it was found that all of thevectors bearing RSV F B3CT and B3TMCT replicated efficiently in vitro tohigh titers that were indistinguishable from those of vector with RSV Fprotein that was unmodified with respect to TM and CT (HEK/GA-opt, FIG.20). Thus, there was no evidence of any reduction in replication invitro due to the increased incorporation of RSV F into the vectorparticle. This is important because efficient vector replication invitro would be essential for efficient vaccine manufacturing andclinical evaluation. It also suggests that the high efficiency ofincorporation will not place a strong selective pressure for mutationsthat silence expression of RSV F.

The intracellular expression of the chimeric forms of RSV F by therB/HPIV3 vectors was examined by Western blotting. This was evaluated inVero cells that were harvested 48 h post-infection (FIG. 21).Interestingly, the B3CT and B3TMCT versions of F were both expressedefficiently, and indeed appeared to be expressed slightly moreefficiently than the native F (i.e., HEK/GA-opt) (FIG. 21A). Inaddition, the DS or DS-Cav1 modifications appeared to further increaseexpression (FIG. 21B), as noted previously (FIG. 13B). These effectsappeared to be additive, since the DS or DS-Cav1 constructs with B3CT orB3TMCT were expressed even more efficiently than those with DS orDS-Cav1 alone. This increased expression would be advantageous forvaccine purposes since it provides a higher level of antigen. As noted,protein engineering and domain swapping have the potential to negativelyaffect the expression, processing, and stability of a glycoprotein, andso the efficient expression of these glycoproteins with DS, DS-Cav1,B3CT, and B3TMCT modifications by this live vector was a property thatcould not have been reliably predicted.

The ability of these constructs to induce syncytium formation in Verocells was assayed. Unexpectedly, RSV F bearing the B3CT substitutionexhibited a hyper-fusogenic phenotype, while that bearing the B3TMCTsubstitution resembled native F (e.g., HEK/GA-opt) in beinghypo-fusogenic (FIG. 22; also see FIG. 3). Upon extended incubation,B3TMCT did induce syncytium formation in the cell monolayer, indicatingit was still functional and thus was conformationally intact. Thesefindings suggested that, between the B3CT and B3TMCT constructs, thelatter would be preferred since extensive syncytium formation andresulting cytopathology might reduce vector production in vitro and invivo by prematurely destroying the cell substrate, and also mightinterfere with the stability of infectivity due to premature triggering.None of the pre-fusion DS forms induced syncytia in the cell monolayers.This was not completely unexpected, since a stabilized version of thepre-fusion F protein should be less able to undergo the massiveconformation changes needed to mediate fusion. These findings suggestthat the pre-fusion DS form of RSV F indeed was substantially stabilizedin the context of vector-infected cells.

Replication of vectors expressing the B3CT and B3TMCT RSV F constructswas examined in hamsters by intranasal infection (FIG. 23). Vectors withF that was non-HEK non-optimized (non-HEK/non-opt) or F that was HEK andGA-optimized (HEK/GA-opt) were somewhat more attenuated in the nasalturbinates compared to empty rB/HPIV3 vector, illustrating theattenuating effect of the insert. Increased attenuation compared to theempty vector was evident by the lower values for virus shedding. It alsowas evident by comparison of the day 3 and day 5 titers: for the emptyvector, these values were comparable, whereas for the vectors bearingRSV F, the day 3 titers were lower than the day 5 titers, indicatingthat these constructs took longer to achieve their maximum titers.) Thevectors expressing B3CT, or B3TMCT, or pre-fusion F (DS) with B3CT(DS/B3CT) or B3TMCT (DS/B3TMCT) were substantially (and in most casessignificantly) more attenuated in the nasal turbinates (FIG. 23A). Thislikely reflects an attenuating effect of the incorporation of the RSV Fprotein into the vector particle: this attenuating effect was notobserved in vitro (FIG. 20). In the lungs, all of the vectors expressingRSV F were substantially more attenuated compared to empty vector (FIG.23B). It is noteworthy that the hyper-fusogenic B3CT construct wassignificantly more attenuated in both the nasal turbinates and the lungsthan the parallel stabilized DS/B3CT construct, suggesting thatincreased fusion indeed was attenuating (i.e., interfered withreplication) in vivo under these conditions. If this was evident in asemi-permissive host such as the hamster, it might be substantially morepronounced in the human host. The non-attenuated wt RSV (A2) controlreplicated to 100- to 1000-fold higher titer in nasal turbinates, and1000 to 10,000-fold higher titer in lungs, compared to the attenuatedvectors.

The immunogenicity of the vectors was determined by analyzing hamstersera for RSV-neutralizing antibodies by a 60% plaque reduction assay inthe presence or absence of added complement (FIGS. 24 A and B,respectively). All of the constructs expressing F protein with B3CT orB3TMCT modifications induced substantial titers of RSV-neutralizingserum antibodies detected in the presence of complement (FIG. 24A). Theconstructs that combined B3CT or B3TMCT with the prefusion DS mutationsgave somewhat higher levels of neutralizing antibodies compared to theparallel constructs without the DS mutations. When assayed in thepresence of complement (FIG. 24A), all of the attenuated vectorconstructs induced lower titers of RSV-neutralizing antibodies comparedto non-attenuated wt RSV, although as noted wt RSV has the advantage ofthe further contribution of the G neutralization antigen and replicated100- to 10,000-fold more efficiently than the attenuated vectors.

In the version of the assay performed without complement (FIG. 24B)three vectors efficiently induced RSV-neutralizing antibodies detectedunder these conditions, namely the one expressing B3TMCT F and the onesexpressing pre-fusion DS F containing the B3CT and B3TMCT modifications.The B3TMCT and DS/B3TMCT constructs induced somewhat more high-qualityRSV-neutralizing serum antibodies than wt RSV, although this differencewas not significant. Nonetheless, this finding was remarkable given thatthe vectors expressed only one of the two RSV neutralizing antigens andreplicated 10- to 10,000-fold less efficiently compared to wt RSV (FIG.22). In contrast to the B3TMCT vectors, B3CT did not induce asignificant antibody response when assayed in the absence of complement(FIG. 24B), even though it was incorporated in the virions at a similarefficiency as the B3TMCT (FIG. 18). Similarly, DS/B3CT also was lessimmunogenic than B3TMCT, DS (FIG. 24B). This indicated that B3TMCT maybe a structurally or antigenically superior form of RSV F compared toB3CT, or it may be that the hyperfusogenic phenotype of B3CT reduced itsexpression and immunogenicity in vivo. These studies clearly showed thatB3TMCT greatly enhanced the immunogenicity of RSV F.

To evaluate the protective efficacy of the vectors, immunized hamsterswere challenged intranasally with 10⁶ pfu of wt RSV at 30 dayspost-immunization (FIG. 25). In agreement with the immunogenicity data,B3CT was less protective in both the nasal turbinates and the lungs.B3TMCT was more protective against RSV challenge, and DS/B3TMCT was themost protective of the vector constructs (although the difference wassmall), with only one immunized hamster showing detectable RSVreplication in the nasal turbinates and all being completely protectedin the lungs, providing protection similar to that conferred by thenon-attenuated wt RSV control (FIG. 24). The comparable level ofprotective efficacy between the DS/B3TMCT construct and wt RSV wasparticularly noteworthy because the latter replicated to 2-4 log titershigher than DS/B3TMCT, expressed both the F and G RSV neutralizationantigens, and expressed all of the RSV proteins as potential antigensfor cellular immunity. This indicates that rB/HPIV3 vector with packagedpre-fusion form of RSV F (DS/B3TMCT) is very highly immunogenic andprotective.

Stability of the rBfHPIV3-RSV-F Constructs in Hamsters.

Give the experience with the genetic instability of MEDI-534 in clinicalstudies (Yang et al 2013 Vaccine 31:2822-2827), a key issue was whetherexpression of the RSV F insert remained stable during replication invivo. The genetic stability of all of the 12 different rB/HPIV3-RSV-Fconstructs that had been analyzed in hamsters in FIGS. 8, 14, and 23 wasassayed. Tissue homogenates of the lungs and nasal turbinates that werecollected on days 3 and 5 following infection were analyzed by afluorescence double-staining plaque assay that can simultaneously detectthe expression of the RSV F protein and the vector proteins in the viralplaques (FIG. 26). In this assay, RSV F expression was detected with anF-specific antibody visualized by red fluorescence, and expression ofPIV3 antigen was detected with an HPIV3-specific antiserum (from rabbitsthat were hyperimmunized with purified HPIV3 virions) visualized bygreen fluorescence. When merged, rB/HPIV3 plaques that maintainedexpression of RSV F appeared yellow while those that have lostexpression of the RSV F insert remained green (FIG. 26). This analysisshowed that the RSV F insert generally was stable during replication inhamsters (FIG. 26). For majority of the samples, all of recoveredviruses in higher dilution wells (in which individual plaques could bediscerned) appeared as yellow plaques and thus expressed RSV F protein.In a subset of other specimens there was sporadic loss of expression ofRSV F in a subset of plaques, resulting in a small percentage of greenplaques (usually <12%). In addition, there was no evidence that loss ofexpression of RSV F increased progressively with time; in other words,there was not a higher frequency of loss of expression on specimens fromday 5 compared to day 3. In a single case, there was a high level ofloss of expression in a single nasal turbinate specimen from day 3 (14%remaining expression, hamster #511 in group #9), whereas the lungspecimen from the same animal had 100% expression. In general, thisindicated that expression of the RSV F insert was substantially stablefor all of the tested constructs. It also showed that none of theconstructs appeared to be disproportionately unstable. Thus, high levelsof expression of RSV F and syncytium formation, or expression ofstabilized forms of RSV, or high levels of incorporation of modified Finto the vector particles that was attenuating for the rB/HPIV3 vector,did not appear to favor the emergence of mutants in which expression ofRSV F was silenced.

The 12 rB/HPIV3 constructs described and evaluated in FIGS. 1-26 werefurther evaluated for possible temperature sensitivity phenotype. Foreach vector, equal aliquots were plagued under methylcellulose at 32,35, 36, 37, 38, 39, and 40° C. (FIG. 27). A reduction in plaqueformation of ≥100-fold was indicative of temperature sensitivity at thattemperature. The empty rB/HPIV3 vector was temperature sensitive (ts) at40° C. (FIG. 27), whereas neither HPIV3 nor BPIV3 is ts at thistemperature. This indicates that chimerization (i.e., the replacement ofthe HPIV3 F and HN genes into the BPIV3 backbone) conferred a slight tsphenotype. Every vector that in addition contained the RSV F insert wasts at 37-38° C., indicating that the presence of the additional geneaugmented the ts phenotype. The constructs that were the most ts wereB3CT, B3TMCT, and DS/B3CT (#7, 8, 12 in FIG. 27). This implied thatpackaging of RSV F into the virus particle augmented this phenotype. Onepossible explanation would be that the presence of RSV F in the vectormade the particle somewhat unstable and susceptible to elevatedtemperature. The remaining construct with efficient packaging of RSV F,namely DS/B3TMCT (construct 13 in FIG. 27), was slightly less ts,suggesting that the hypo-fusogenic phenotypes associated with DS andB3TMCT ameliorated the instability to some extent. Taken together, thesefindings provide a means to confer attenuation or to mitigateattenuation, depending on the construct design, and in any event provideinformation important in designing vaccine viruses.

Evaluation of Selected rB/HPIV3-RSV-F Constructs in Rhesus Macaques.

To further investigate the effects of the “DS” and “B3TMCT” mutations onvector replication, immunogenicity, and protective efficacy, twocandidates (HEK/GA-opt/DS and HEK/GA-opt/DS/B3TMCT) were evaluated forreplication and immunogenicity in rhesus macaques (FIG. 28). The B/HPIV3vector with unmodified RSV F (non-HEK/non-opt) was included as abaseline control for comparison. A limited number of constructs wereevaluated given the expense and ethical consideration of studies inprimates. Monkeys were immunized with a total of 2×10⁶ TCID₅₀ of eachrBHPIV3 vector by the combined IN and intratracheal (IT) routes.Nasopharyngeal swabs and tracheal lavage samples were collected onindicated days (FIG. 29) to monitor virus shedding as a measure ofreplication. Sera were collected on day 0, 14, 21, and 28 days. Allanimals were challenged on day 28 with wt RSV IN and IT, with 10⁶ pfuper site, and serum samples were collected on days 35 and 56.

The non-HEK/non-opt and HEK/GA-opt/DS viruses replicated to peak titersof approximately 10⁵ and 10³ TCID₅₀ units per ml in the upper and lowerrespiratory tracts (FIGS. 29A and B, respectively). In contrast, theHEK/GA-opt/DS/B3TMCT construct was dramatically more attenuated in boththe upper and lower respiratory tracts of rhesus monkeys (FIGS. 29 A andB). This indicated that B3TMCT conferred substantial attenuation to therB/HPIV3 construct, whereas the DS mutations did not appear to confersignificant attenuation. Thus, the mild tendency of the B3TMCTmodification to confer attenuation in hamsters (FIG. 23) wassubstantially greater in non-human primates.

As noted, sera were collected on days 0, 14, 21, 28, 35, and 56. Serumantibodies specific to the rB/HPIV3 vector were analyzed by a 60% plaquereduction assay against HPIV3 (FIG. 30). This showed that thevector-specific neutralizing serum antibody response to the highlyattenuated HEK/GA-opt/DS/B3TMCT construct was slower and somewhatreduced compared to the non-HEK/non-opt and HEK/GA-opt/DS constructs.This is consistent with the expectation that reduced antigenic loadwould reduce immunogenicity. After 28 days the titers became moresimilar and, as expected, there was no boost in vector-specific immunityfrom the RSV challenge on day 28 (since RSV did not contain any vectorantigens).

The RSV-specific neutralizing serum antibody responses were quantifiedby a plaque reduction assay performed in the presence and absence ofcomplement (FIGS. 31 and 32, respectively). The assay in the presence ofcomplement showed that RSV-neutralizing serum antibodies were inducedmore rapidly and to significantly higher titer in response to theHEK/GA-opt/DS/B3TMCT construct than for the other two constructs (FIG.31). This greater induction of RSV-specific neutralizing serumantibodies was noteworthy and surprising since this construct was muchmore highly restricted in replication (FIG. 29). When assayed previouslyin hamsters (FIG. 24), this construct had given an apparent increasecompared to HEK/GA-opt/DS that was not statistically significant: thegreater increase observed in non-human primates is consistent with theidea that the hamster model is a less sensitive model for these humanand bovine/human viruses. For example, The greater induction ofRSV-neutralizing serum antibodies in the rhesus macaques in response tothe HEK/GA-opt/DS/B3TMCT construct also was observed when the assay wasperformed in the absence of complement to measure high-qualityantibodies on day 28: indeed, the difference in titer for this constructversus the other two constructs was substantially greater than what wasobserved in the presence of complement (FIG. 32). Taken together, theseresults indicated that the DS mutation did not substantially increasethe quantity of RSV-neutralizing serum antibodies (FIG. 31) but didincrease the quality (FIG. 32), while the further addition of the B3TMCTmodification dramatically increased both the quantity (FIG. 31) andquality (FIG. 32) of the RSV-neutralizing serum antibodies.

The RSV challenge virus administered on day 28 was completely restrictedby all vectors and no infectious challenge RSV could be recovered fromthe nasopharyngeal swabs and tracheal lavage samples from any animal,and therefore this experiment did not provide further information on thecomparative properties of these viruses. Complete protection againstshort-term RSV challenge in experimental animals is sometimes observedbecause the semi-permissive nature of RSV replication in these modelsfacilitates restriction of replication. The RSV-specific antibodyresponses continued to increase following day 28, but it is not clearwhether this response was due to the primary infection or the challenge.

The fluorescence double-staining plaque assay was used to analyze virusrecovered from the rhesus macaques on days 4, 5, and 6, which was thetime of peak shedding, for the expression of RSV F protein. A summary ofthe data is shown in FIG. 33. This analysis showed that the RSV Finserts generally were stable during replication in monkeys. The resultswere very similar to those shown in FIG. 26 for the hamster study. Formajority of the samples from the rhesus monkeys, nearly all of recoveredviruses appeared as yellow plaques and thus expressed RSV F protein.There was sporadic loss of RSV F expression in some specimens, typically<10% of recovered plaques in a given specimen. There was no evidencethat loss of expression increased significantly with time, and sometimesevidence of loss of expression was observed at an early time point butnot at a later time point from the same animal. There also was noevidence that a particular construct was associated withdisproportionately greater loss of RSV F expression versus anotherconstruct. For example, even though the expression of HEK/GA-opt/B3TMCTwas highly attenuating for the rB/HPIV3 vector, there was no evidence ofincreased selection of virus in which expression of RSV F was lost.

A rB/HPIV3 vector expressing the ectodomain of RSV F (amino acids 1-513,lacking the TM and CT domains) with GenScript (GS) optimization, andcontaining the HEK assignments and the DS-Cav1 modifications was alsogenerated. The ectodomain was fused to a 4-amino acid linked followed bya trimer-stabilizing foldon sequence at its C-terminus, i.e. construct#19 (HEK/GS-opt/DS-Cav1/[1-513] Foldon). This form of pre-fusion RSV Fwas previously evaluated as subunit vaccine in mice and rhesus monkeys(McLellan, et al. 2013. Science 342:592-598). This RSV F protein shouldbe expressed as a partially secreted form, resembling construct #8(HEK/GA-opt/Ecto), but with the improvements of more efficienttranslation due to the GS optimization, better immunogenicity due to theDS-Cav1 modifications, and greater trimer stability due to the foldonstabilization domain. This construct can be used to compare howimmunogenic the secreted DS-Cav1 form is compared with the membraneanchored form, i.e. #16 (HEK/GS-opt/DS-Cav1) and the virion-incorporatedform, i.e. #18 (HEK/GS-opt/DS-Cav1/B3TMCT).

Points from this study: (1) The hamster model, while convenient, may besomewhat insensitive to changes in replication, expression, andimmunogenicity with the various constructs, and it may be that theeffects associated with these constructs, such as attenuation andimmunogenicity, would be substantially greater in the human host forwhich these vaccine constructs are intended. For example, while thepresence of the B3TMCT modification in the HEK/GA-opt/DS/B3TMCTconstruct conferred only a modest increase in attenuation in the hamstermodel (FIG. 23), it was substantially more attenuating in rhesus macaque(FIG. 29), which is more closely related to the authentic human host.Furthermore, the difference in immunogenicity for RSV-neutralizingantibodies between HEK/GA-opt/DS/B3TMCT and the other constructs wassubstantially greater in rhesus macaques (FIG. 31) than in hamsters(FIG. 24), even though that construct was substantially more attenuatedin rhesus macaques (FIG. 29). Thus, the inherent immunogenicity per pfuof HEK/GA-opt/DS/B3TMCT appeared to be much greater in rhesus macaquesthan in hamsters, and thus might similarly be greater in humans. Thisapparent insensitivity in the hamster may explain, for example, why thehamster model did not reliably provide a difference in immunogenicityassociated with a 16-fold increase in RSV F expression (FIG. 9). It alsoshould be noted that codon-optimization for human use might not providecomparable increases in expression in hamsters as compared to primatecells (and the human vaccinee), which might contribute to reducedimmunogenicity in the hamster model compared to primates. (2) Anothertheme was the desirability to reduce the amount of syncytium formationinduced by RSV F. In vitro, syncytium formation did not appear torestrict replication in monolayer cultures, although it is possible thatit might become a factor in microcarrier cell culture systems used formanufacture, and so it seems prudent to control syncytium formation.With most of the constructs, the HEK assignments were present, whichstrongly suppressed syncytium formation. The DS and DS-Cav1 constructs,like the HEK assignments, also strongly suppressed syncytium formationand thus provided an unexpected benefit. One construct that wasassociated with up-regulated syncytium formation, namelyHEK/GA-opt/B3CT, did exhibit reduced replication (FIG. 23) andimmunogenicity (FIG. 24) in hamsters, giving an indication thatincreased syncytium formation can be deleterious in vivo. This might bemore pronounced in a primate host. Therefore, the combination of GS-opt,HEK, DS or DS-Cav1, and B3TMCT was identified as a combination thatwould give the highest level of expression of RSV F (due to HEK plusGS-opt, plus stabilization of the pre-fusion F protein that appeared toprovide increased F protein accumulation) in the context of twosuppressors of syncytium formation (HEK and DS-Cav1), and in thepresence of packaging signals that were not hyper-fusogenic (B3TMCT).(3) Two features (namely B3TMCT and the DS mutations) independently wereassociated with substantial induction of high quality RSV-neutralizingserum antibodies. The rhesus macaque study suggested that the B3TMCT wasthe more important of these two factors in a primate host, relevant toanticipated vaccine performance in humans (FIG. 32). Importantly, theB3TMCT modification also dramatically increased the quantity (FIG. 31)of the RSV-neutralizing serum antibody response. (4) Unexpectedly andfortuitously, the features that improved the expression and packaging ofRSV F did not appear to confer a significant selective pressure for lossof expression of RSV F, when evaluated by a dual immunofluorescenceassay. As already noted, loss of expression of the RSV F insert was aproblem with MEDI-534, but a number of features were employed in thepresent study to down-regulate fusion that were not employed inMEDI-534, and this may have played a major role in stabilizing the RSV Finsert. In addition, by evaluating two overlapping sets of packagingsignals, it was possible to identify and avoid one that washyperfusogenic, and to identify and choose one that had substantiallyreduced fusion. (5) The B3TMCT modification in particular emerged as animportant factor in increasing immunogenicity. The HEK, GA-opt orGS-opt, and DS or DS-Cav1 modifications emerged as secondaryimprovements. B3TMCT had the effect of strongly attenuating the vectorin rhesus macaques, as noted. The use of this modification in thecontext of the highly attenuated rB/HPIV3 vector resulted in a vectorthat appeared to be substantially over-attenuated. However, usingreverse genetics, the B3TMCT F protein (with HEK, plus GA- or GS-opt,plus DS or DS-Cav1 modifications, as desired) can be combined with aless-attenuated vector backbone, such as wild type HPIV1, 2, or 3 orversions bearing one or more known, stabilized attenuating mutations, tocreate a construct that is less attenuated. Since the B3TMCT constructin rB/HPIV3 was very highly immunogenic despite being veryover-attenuated, a construct expressing this protein that replicates 10-to 100-fold better should be suitably attenuated and substantially moreimmunogenic.

Additional Assays with Recombinant rBfHPIV3 Vectors Expressing ModifiedVersions of the RSV F ORF and Protein.

Additional assays were preformed to evaluate: (i) GS-opt versions ofconstructs including the DS prefusion stabilization mutations and theB3TMCT packaging signal, and (ii) the DS-Cav1 prefusion stabilizationmutations, which include the two cavity-filling mutations S190F andV270L combined with the DS mutations. The F proteins assayed alsocontain the two HEK amino acid assignments that result in an amino acidsequence identical to that of an early passage (called HEK-7) of the A2strain, as described above. These assays show that:

1. DS-Cav1 and B3TMCT independently confer the ability to inducesignificant levels of complement-independent RSV-neutralizingantibodies, which are considered to be the most relevant for in vivoprotection.

2. The combination of DS-Cav1 plus B3TMCT gives a further increase inimmunogenicity.

3. The two most immunogenic constructs were HEK/GA-opt/DS-Cav1/B3TMCT(FIG. 53, group #7) and HEK/GS-opt/DS-Cav1/B3TMCT (group #10), whichdiffer only in the source of codon optimization, with GS-opt appearingto be the most immunogenic.

4. Although HEK/GA-opt/DS-Cav1/B3TMCT (group #7) andHEK/GS-opt/DS-Cav1/B3TMCT (FIG. 53, group #10) were not statisticallydistinguishable in a pair-wise comparison, the latter was significantlymore immunogenic than wt RSV. These findings show thatHEK/GS-opt/DS-Cav1/B3TMCT (group #10) was the most immunogenic constructin the hamster model, particularly for highly-efficient neutralizingantibodies detected without the need to add complement (FIG. 53), andthus the further modification with GS-opt and DS-Cav1 appeared toincrease immunogenicity. This also was the most protective construct inthe hamster challenge study.

5. The propensity for the rB/HPIV3 vector to acquire mutationsconferring a large-plaque phenotype and attenuation was essentiallyeliminated by three nucleotide and two amino acid mutations in thevector HN protein.

6. The HEK/GS-opt/DS-Cav1/B3TMCT insert was expressed from the firstgene position (pre-N), resulting in a construct that replicatedefficiently in Vero cells, could be obtained in a preparation with ahigh percentage of RSV F expression, and efficiently expressed the RSV Fprotein.

Summary of Animal Studies.

FIG. 35 indicates the constructs that have been evaluated in twodifferent studies in hamsters and two different studies in rhesusmonkeys, as indicated: the “1^(st) hamster study” encompasses FIGS. 8-9,14-16, and 23-26; the “1^(st) NHP study” encompasses FIGS. 29-33; the“2^(nd) hamster study” encompasses FIGS. 51-54; the “2^(nd) NHP study”encompasses FIGS. 55 and 57.

Multi-Cycle Replication In Vitro of GA-Opt Viruses.

FIGS. 46 and 47 illustrate multi-cycle replication of theHEK/GA-opt/DS-Cav1 construct on its own or with the further addition ofthe B3TMCT packaging signal (constructs #13 and 15 in FIG. 35), comparedto the empty vector. This was done in African Green monkey kidney Verocells (FIG. 46), which is the cell substrate used for vaccinemanufacture, and in rhesus monkey kidney LLC-MK2 cells (B), which is acommon laboratory cell line that, unlike Vero cells, typically isinduced by virus infection to express type I interferons. Thisexperiment showed that the two DS-Cav1-containing constructs (with orwithout B3TMCT) replicated efficiently and similarly to each other, andwere modestly attenuated compared to the empty vector. Despite thismodest attenuation, both DS-Cav1-containing constructs replicated totiters higher than 10⁷ TCID₅₀/ml. This pattern of modest attenuation isvery similar to results obtained with other rB/HPIV3 vectors expressingdifferent versions of RSV F (e.g. FIGS. 7, 12, and 20). Thus, theseresults showed that rB/HPIV3 bearing these modified inserts replicatedin an efficient manner that is fully satisfactory for vaccinemanufacture.

Multi-Cycle Replication In Vitro of GS-Opt Viruses.

FIG. 48 illustrates multi-cycle replication of the HEK/GS-opt backbone(construct #5 in FIG. 35) compared with versions with the furtheradditions of DS-Cav1 (#16), the combination of DS-Cav1/B3TMCT (#18), andthe combination of DS-Cav1, ectodomain 1-513, and a C-terminal “foldon”domain to promote ectodomain oligomerization (#19), with empty vectorfor comparison. Evaluation in Vero (FIG. 48A) and LLC-MK2 (B) cellsshowed that the vectors expressing the various forms of RSV F replicatedwith very similar kinetics and yield, and were modestly attenuatedcompared to the empty vector. They all replicated to titer higher than10⁷ TCID₅₀/mL in cell culture. These results showed that rB/HPIV3bearing these modified inserts replicated in an efficient manner that isfully satisfactory for vaccine manufacture.

Multi-Cycle Replication In Vitro of GA- and GS-Opt Viruses.

FIG. 49 illustrates multi-cycle replication of pairs of constructs thatdiffer in being GA-optimized or GS-optimized: specifically:HEK/GS-opt/DS-Cav1 versus HEK/GA-opt/DS-Cav1 (top panels, constructs #16and 13 respectively from FIG. 35), and HEK/GS-opt/DS-Cav1/B3TMCT versusHEK/GA-opt/DS-Cav1/B3TMCT (bottom panels, constructs #18 and 15respectively from FIG. 35). The two pairs of constructs were eachevaluated in Vero (FIG. 49A, C) and LLC-MK2 (B, D) cells. The GS-optconstructs sometimes replicated marginally more efficiently than theGA-opt constructs (e.g. FIGS. 49C and 49D), especially during the first3-4 days of incubation.

Expression of RSV F In Vitro.

FIG. 50 illustrates expression of RSV F and vector proteins by rB/HPIV3constructs in Vero and LLC-MK2 cells. This compares expression byHEK/GA-opt (FIG. 50, lane 2; FIG. 35, construct #3) versus HEK/GS-opt(FIG. 50, lane 3; FIG. 35, construct 5): consistent with resultsdescribed in Example 1 (FIG. 5), the GS-optimization resulted insomewhat greater expression of RSV F protein. The HEK/GS-opt constructalso was compared to versions that, in addition, contained DS-Cav1 (FIG.50, lane 3; FIG. 35, construct #16), or DS-Cav1/B3TMCT (FIG. 50, lane 5;FIG. 35, construct #18), or DS-Cav1/(1-513)Foldon (FIG. 50, lane 8; FIG.35, construct #19). Also included for comparison were cells infectedwith wt RSV (FIG. 50, lane 6) or mock-infected (lane 7). The resultsshowed that each of the GS-opt constructs directed efficient expressionof RSV F, and indeed expressed much more RSV F than did wt RSV (FIG. 50,lane 6). Cells infected with the three GS-opt constructs encodingfull-length DS-Cav1 F protein (FIG. 50, lanes 4, 5, and 8) had asomewhat greater accumulation of the F0 precursor of RSV F protein,suggesting that its prefusion stabilization may have marginally reducedthe efficiency of cleavage. In general, however, each of the constructswas very efficient in expressing the RSV F protein. The prefusion Foldonconstruct, HEK/GS-opt/DS-Cav1/(1-513)Foldon (FIG. 50, lane 8), was onlypartly secreted into the medium (FIG. 50A, lower panel), and thesecreted form was entirely the cleaved F₁ chain (FIG.4A, lower panel,lane 8), whereas all of the cell-associated form was uncleaved F₀ (FIG.4A, upper panel, B and C, lane 8). None of other tested forms of RSV Fwas detected in the medium supernatant, indicating they were notsecreted. The inefficient cleavage and secretion of theDS-Cav1/(1-513)Foldon construct was unexpected, since this trimerizationdomain had been successfully used previously to prepare purified Fprotein (McLellan et al Science 2013 Nov. 1; 342(6158):592-8. doi:10.1126/science.1243283). This illustrates that expression of foreignproteins by vectored constructs can be unpredictable, whereas thedetailed evaluation of multiple constructs herein provides comprehensiveevaluation leading to the identification of a number of suitable,successful constructs.

Hamster Studies.

A number of GA-opt and GS-opt constructs that contained the furtheradditions of B3TMCT, DS, and DS-Cav1 (the constructs are identified inFIG. 35 as the “2^(nd) hamster study”) were evaluated in hamsters forefficiency of replication (FIG. 51), stability of expression of RSV Fprotein, stimulation of RSV-neutralizing serum antibodies (FIGS. 52 and53), and protective efficacy against RSV challenge (FIG. 54).

Replication in Hamsters.

GA-opt and GS-opt constructs were evaluated for replication in the upper(nasal turbinates) and lower (lungs) respiratory tract of hamsters (FIG.51). In the nasal turbinates, HEK/GA-opt/B3TMCT (group #5) andHEK/GA-opt/DS/B3TMCT (group #6) were more restricted than others. In thelungs, GA-opt constructs with B3TMCT (HEK/GA-opt/B3TMCT, group #5,HEK/GA-opt/DS/B3TMCT, group #6, and HEK/GA-opt/DS-Cav1/B3TMCT, group #7)were more restricted. Similarly, GS-opt constructs with B3TMCT, i.e.,HEK/GS-opt/DS-Cav1/B3TMCT (group #10) was also more attenuated thanHEK/GS-opt/DS-Cav1 (group #9). These observations suggested that B3TMCTincreases the level of attenuation. In addition, GA-opt constructsappeared to be more attenuated than GS-opt constructs. For example,GS-opt constructs of DS-Cav1 and DS-Cav1/B3TMCT, i.e.,HEK/GS-opt/DS-Cav1 (group #9) and HEK/GS-opt/DS-Cav1/B3TMCT (group #10)replicated to mean peak titers of 5.0 and 4.2 Log₁₀ TCID₅₀/g in the LRT,which was higher than the mean titers of equivalent GA-opt constructs,i.e. 4.0 and 3.4 Log₁₀ TCID₅₀/g (groups #4 and 7). This suggested thatGS-opt RSV F inserts were less attenuating than GA-opt RSV F.

Stability of Expression of RSV F Protein.

The stability of RSV F expression by rB/HPIV3 vectors during theirreplication in vivo was evaluated with double-staining plaque assay byanalyzing nasal turbinate and lung samples of immunized hamstersharvested on day 5 post-immunization. Most of samples (92 out of 107)had more than 90% of replicated vectors still expressing RSV F; 6 out of107 had 89-80% vectors expressing RSV F; 9 out of 107 had less than 79%of replicated vectors expressing RSV F; only 7 samples had >50% ofvectors losing RSV F expression. Among these seven samples with >50%vectors losing RSV F expression, four were GA-opt constructs, three wereGS-opt constructs. There was no evidence that GA-opt, or GS-opt, or DS,or DS-Cav1, or TMCT were associated with any particular increase ininstability. It is likely that the varying levels of instability amongindividual preparations reflect sporadic mutations that are largelyindependent of the specific construct, and thus evaluation of severalindependent preparations of each construct likely would identify one ormore with a very high percentage of expression of RSV F protein.

Titers of RSV-Neutralizing Serum Antibodies.

RSV neutralizing serum antibody titers were determined by RSVneutralization assays with added guinea pig complement (FIG. 52) or inthe absence of complement (FIG. 53). The assay performed with addedcomplement is commonly used for RSV and HPIV3 neutralization assaysbecause it allows sensitive detection of virus-specific antibodies,since complement can confer viral-lysis and steric-hindrancecapabilities to antibodies that otherwise might not be neutralizing invitro (Yoder et al J Med Virol 72:688-694, 2004). In contrast,antibodies that neutralize RSV in vitro in the absence of complementhave been suggested to be the most relevant for protection (Yoder et alJ Med Virol 72:688-694, 2004), and in prior studies have associated witha higher level of protection against RSV challenge (Liang et al J Virol89:9499-9510, 2015). Antibodies that neutralize in vitro independent ofadded complement are considered to be “high quality” and to beindicative of qualitatively superior immunogenicity.

In the complement-dependent assay (FIG. 52), all rB/HPIV3 vectorsexpressing RSV F induced high titers of RSV neutralizing antibodiesexcept for the poorly immunogenic HEK/GS-opt/DS-Cav/(1-513)Foldonconstruct. GS-opt constructs induced higher titers of RSV serumneutralizing antibodies than their counterparts of GA-opt constructs(11.0 Log₂ PRNT₆₀ for HEK/GS-opt/DS-Cav1, group #9 versus 10.2 Log₂PRNT₆₀ for HEK/GA-opt/DS-Cav1 group #4; 11.8 Log₂ PRNT₆₀ forHEK/GS-opt/DS-cav1/B3TMCT, group #10 versus 10.4 Log₂ PRNT₆₀ forHEK/GA-opt/DS-Cav1/B3TMCT, group #7). In the complement-dependent assay(FIG. 52), it was noteworthy that the three constructs that werestatistically as immunogenic as wt RSV (the gold standard) were theGS-opt constructs HEK/GS-opt (group #8), HEK/GS-opt/DS-Cav1 (group #9),and HEK/GS-opt/DS-Cav1/B3TMCT (group #10).

In the complement-independent assay (FIG. 53), vectors expressing nativeforms of RSV F (Non-HEK/non-opt and HEK/GS-opt) did not inducedetectable RSV neutralizing antibodies, confirming and extending resultsfrom Example 1. Prefusion stabilizing mutations (DS, DS-Cav1) and thepackaging signal B3TMCT independently increased immunogenicity for highquality RSV-neutralizing antibodies (e.g., constructs with DS and/orCav1 in the absence of B3TMCT are exemplified by groups #3, 4, and 9,whereas a construct with B3TMCT in the absence of DS/Cav-1 isexemplified by group #5). The combination of prefusion stabilizingmutations plus B3TMCT had additive improvement in immunogenicity, whichwas observed with GA-opt as well as GS-opt constructs (e.g., groups 6,7, and 10). In the complement-independent assay, theHEK/GS-opt/DS-Cav1/B3TMCT construct (group #10) induced significantlyhigher titers of RSV-neutralizing serum antibodies (7.6 Log₂ PRNT₆₀)than that induced by any of the other vectors except for its GA-optcounterpart HEK/GA-opt/DS-Cav1/B3TMCT (group #7) which induced a lowertiter (6.8 Log₂ PRNT₆₀) but was not significantly lower. However, theantibody titer induced by HEK/GS-opt/DS-Cav1/B3TMCT construct (group#10), but not HEK/GA-opt/DS-Cav1/B3TMCT (group #7) was significantlyhigher than that of wt RSV (group 12), and therefore theHEK/GS-opt/DS-Cav1/B3TMCT construct (group #10) was the most immunogenicamong the vectors and wt RSV for high quality RSV-neutralizingantibodies.

RSV Challenge.

The immunized hamsters were challenged IN with wt RSV to evaluateprotective efficacy (FIG. 54). As shown in FIG. 54, all of the RSVF-expressing vectors, except for the DS-Cav1/(1-513)Foldon construct,induced significant protection in the URT (FIG. 54A) and LRT (FIG. 54B).The lack of protection by DS-Cav1/(1-513)Foldon was in line with itspoor immunogenicity shown in the RSV serum neutralization assays (FIGS.52 and 53). Compared with the sterile immunity conferred by wt RSV, theHEK/GS-opt/DS-Cav1/B3TMCT conferred the greatest protection among alltested vectors and was nearly equivalent to wt RSV: only one hamsterimmunized by this vector had detectable wt RSV, and only at very lowlevel in the nasal turbinates. The protective efficacy ofHEK/GS-opt/DS-Cav1/B3TMCT (group #10) was statisticallyindistinguishable from that of wt RSV (group #12) in the nasalturbinates, whereas HEK/GA-opt/DS-Cav1/B3TMCT (group #7) wassignificantly less protective, supporting the idea that the formerconstruct is the most immunogenic construct. This equivalence inprotection to wt RSV is remarkable because wt RSV expresses twoneutralization antigens, G and F, and also expresses all of the viralproteins as potential antigens for cellular immunity, whereas thevectors express only the RSV F protein. Cellular immunity has been shownto confer potent protection in RSV challenge studies in rodents (e.g.,Connors et al J Virol 66:1277-1281 1992).

Evaluation in Rhesus Monkeys.

Three vectors with greatest immunogenicity in hamsters were selected toevaluate their replication and immunogenicity in rhesus monkeys (FIG.55). These constructs included HEK/GA-opt/DS/B3TMCT (the construct atthe top of FIG. 55), which was identified as particularly effective inthe 1^(st) NHP study (FIGS. 28-32, in Example 1). The second constructwas HEK/GA-opt/DS-Cav1/B3TMCT, which is the same as the first constructexcept that it has DS-Cav1 instead of DS. The third construct wasHEK/GS-opt/DS-Cav1/B3TMCT, which differs from the previous construct inhaving GS-opt instead of GA-opt. These latter two constructs induced thehighest titers of “high quality” RSV-neutralizing serum antibodies inFIG. 53.

Replication in Rhesus Monkeys.

The parallel constructs with GA-opt and GS-opt versions ofHEK/DS-Cav1/B3TMCT replicated in similar kinetics in the URT, as sampledby nasopharyngeal swabs (FIG. 56A). However, this equivalency was notobserved in the LRT, as sampled by tracheal lavage: the GS-opt versionwas less attenuated than GA-opt version (FIG. 56B). These results areconsistent with what was observed in hamsters in FIG. 51. With regard tothe comparison of parallel constructs with DS versus DS-Cav1(HEK/GA-opt/DS/B3TMCT versus HEK/GA-opt/DS-Cav1/B3TMCT), the formervirus was more attenuated in the URT (FIG. 56A); but they replicated atsimilar efficiency in the LRT (FIG. 56B). These results also areconsistent with what was observed in hamsters in FIG. 51. It isnoticeable that the HEK/GA-opt/DS/B3TMCT replicated to significantlyhigher titers (˜10-fold higher) in both URT and LRT in this study,compared to the same construct tested in the 1^(st) NHP study (FIG. 29,Example 1). The monkeys in the previous study (FIG. 29) were 6 yearsolder and 2-3 times bigger in weight than monkeys used in FIG. 56. Itmay be that replication of these viruses is more efficient in youngermonkeys, or the difference might reflect variability between these twoexperiments.

RSV-Neutralizing Serum Antibodies.

Although the three vectors in FIG. 56 replicated at slightly differentefficiencies in rhesus monkeys, they induced comparably high levels ofRSV-neutralizing serum antibodies determined by complement-dependent(FIG. 57A) and complement-independent (FIG. 57B) assays.

Insertion of RSV F at the First Gene Position.

All of the previous B/HPIV3-RSV-F constructs in Examples 1 and thepresent Example involved an RSV gene inserted in the second geneposition, between the vector N and P genes. Insertion of unmodified RSVF at the first position was not associated with any evident problems ofimpaired growth or reduced stability of expression of RSV F protein.Whether an optimized, engineered form of RSV F could be efficiently andstably expressed from the first gene position was investigated.Specifically, the HEK/GS-opt/DS-Cav1/B3TMCT version of RSV F wasinserted into the pre-N position (FIG. 58A).

Identification and Modification of Two Amino Acid Assignments in HN thatConferred Phenotypic Instability of the Vector.

The rB/HPIV3 vector was previously noted to exhibit phenotypicinstability upon passage in vitro (Liang et al J Virol 88:4237-4250).Specifically, a substantial proportion of vector acquired a large-plaquephenotype during passage. This occurred with different inserts as wellas with empty vector, indicating that it was a property of the vectoralone and not specific to the foreign gene. Whole-genome sequenceanalysis of six cloned large-plaque viruses showed that each hadacquired an H552Q missense mutation in HN, as well as one of threedifferent missense mutations in F, and in some cases one or two missensemutations in L (Liang et al J Virol 88:4237-4250). Partial sequencing of11 additional large-plaque clones showed that seven of these containedthe H552Q mutation in HN, while the other four each contained one offour other missense mutation in HN (N240K, P241L, R242K, or F558L).Comparison with the crystal structure of the HPIV3 HN protein (Lawrenceet al J Mol Biol 335:1343-1357 2004) indicated that all of these HNmutations are located in the dimer interface of the HN globular head. Inaddition, the H552Q mutation had previously been described in an HPIV3variant that had been selected by growth on neuraminidase-treated cells,and which had a large-plaque phenotype, had higher avidity to sialicacid-containing receptor, had enhanced triggering of F protein, and wasattenuating in rodents (Moscona et al J Virol 67:6463-6468; Porotto etal J Virol 81:3216-3228; Palermo et al J Virol 83:6900-6908). Thissuggested that the adventitious mutations in the HPIV3 HN gene in therB/HIPIV3 vector were similar and probably were selected for becausethey increased the binding affinity of the rB/HPIV3 vector for Verocells. However, the observation by others (Moscona et al J Virol67:6463-6468; Porotto et al J Virol 81:3216-3228; Palermo et al J Virol83:6900-6908) that the large-plaque phenotype was associated withsubstantial attenuation in vivo would be disadvantageous for the presentvectors because this likely would lead to over-attenuation. The HPIV3reverse genetic system contained two mutations in the HN gene: there wasan adventitious C7589T nucleotide mutation (relative to the completeantigenome sequence) in the cDNA clone leading to a T263I missensemutation, and C7913A and A7915T mutations leading to a P370T mutationthat had been purposefully introduced as a marker (Durbin et al Virology235:323-332, 1997). This latter missense mutation had been designed toablate an epitope recognized by two available HPIV3-neutralizingmonoclonal antibodies (MAbs 423/6 and 170/7). These mutations wererestored to their wild-type assignments, namely 7593C (263T) and7913C+7915A (370P) (FIG. 58B). Remarkably, these changes preventedacquisition of the large-plaque phenotype during passage in Vero cells(not shown). Therefore, further vector constructs bearing the HPIV3 HNgene (including but not limited to rB/HPIV3- and HPIV3-based vectors)preferably should contain these assignments. For example, the I263T andT370P mutations were incorporated into theHEK/GS-opt/DS-Cav1/B3TMCT/pre-N construct shown in FIG. 58A. All vectorsof interest that bear the HPIV3 HN gene will be modified to contain the263T and 370P assignments.

Virus Recovery.

The rB/HPIV3 vector with HEK/GS-opt/DS-Cav1/B3TMCT inserted in the pre-Nposition (FIG. 58A) was recovered and grew to titers up to 8.2 log₁₀TCID₅₀ mL, and thus had little or no growth restriction with regard toviral yield, which is important for vaccine manufacture. The plaque sizeof this virus was generally smaller than the same version of RSV Finserted in the second position (not shown), indicating that there was amodest restriction on growth that was not evident in the virus yield.

Stability of Expression of RSV F Protein.

Two preparations of viruses were analyzed by a double-staining plaqueassay, to evaluate stability of expression of the RSV F protein.Double-staining plaque phenotype of two independent rescued virus pools,designated CL20a and CL24a, were performed. Plaque assay was carried outon Vero monolayer in 24-well plates infected with 10-fold seriallydiluted virus. Infected monolayers were overlaid with medium containing0.8% methylcellulose and incubated at 32° C. for 6 days. After fixing inice-cold 80% methanol, the monolayers were incubated with a mixture ofthree mouse monoclonal antibodies against RSV F (1129, 1109, 1243) and arabbit anti-HPIV3 hyperimmune serum, followed by incubation with IRDye680 (red, detecting RSV F) conjugated goat anti-mouse and IRDye 800(green, detecting HPIV3) conjugated goat anti-rabbit antibodies.

One preparation was stable after passaging in cell culture, whereas asecond preparation had green staining in ˜40% of viruses and thus hadevidence of loss of RSV F expression. These results indicated that lossof RSV F expression can occur and can be amplified in the viruspreparation, but it appeared to be sporadic and careful monitoring canidentify preparations with a high proportion of expression.

Intracellular Protein Expression.

Expression of RSV F from the pre-N position was analyzed in Vero (FIG.59A) and LLC-MK2 (FIG. 59B) cells. In Vero cells,HEK/GS-opt/DS-Cav1/B3TMCT version of RSV F was expressed as F₁ and F₀chains (FIG. 59A), and the total expression from the pre-N position wascomparable to that expressed from the N-P position. But in LLC-MK2 cells(FIG. 59B), this version of RSV F was predominantly expressed as F₀chain, and expression from pre-N was substantially more efficient thanthat from N-P position (FIG. 59B, lane 3, 4, 5). In both types of cells,this version of RSV F was expressed at a much higher level thanunmodified RSV F (Non-HEK/non-opt, FIG. 59A, 59B, lanes 3 and 4 versuslane 6).

Example 2 Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressingthe Fusion F Glycoprotein of Human Respiratory Syncytial Virus (RSV) asa Bivalent HPIV1/RSV Vaccine

This example illustrates the development and pre-clinical evaluation ofa live attenuated rHPIV1 vectored RSV vaccine expressing RSV F antigenfrom three genome positions of the two attenuated rHPIV1 backbones.Pre-clinical evaluation in hamsters indicated that the rHPIV1C^(Δ170-)F1 vector, bearing attenuating deletion mutation (C^(Δ170)) inthe P/C gene and expressing RSV F from the pre-N position wassufficiently attenuated, stable and immunogenic against RSV and HPIV1and provided significant protection against RSV challenge infection.This study demonstrated that rHPIV1 could be used as an RSV vaccinevector to achieve bivalent protection against two major childhooddiseases.

Introduction.

Compared to an RSV vaccine comprising an attenuated strain of RSV, anRSV vaccine comprising a live attenuated HPIV vector (such as onedeveloped from HPIV1) expressing the RSV F protein offers severaladvantages. One advantage is that it provides a bivalent vaccine againstRSV and the HPIV serotype used as a vector. This is important because,as noted, the HPIVs also are important, uncontrolled agents of pediatricrespiratory tract disease, with characteristics of epidemiology andpathogenesis that overlap those of RSV. Thus, a combined HPIV/RSVvaccine is a logical combination that would broaden the coverage againstpediatric respiratory tract disease. In addition, RSV infectivity isnotorious for being prone to instability during handling, whichcomplicates vaccine development, manufacture, and delivery. The HPIVsare substantially more stable, which may be critical for extending RSVvaccines to developing countries where their need is the greatest. RSVgrown in vitro often forms long filaments that complicate manufacture,whereas the HPIVs form smaller spherical particles. It also may be thatRSV is inherently more pathogenic and even possibly immunosuppressivecompared to the HPIVs, which would be another advantage of anHPIV-vectored RSV vaccine. It has also been have found that, in rodents,the use of an HPIV-vectored vaccine as a boost administered subsequentto a live attenuated RSV strain was more immunogenic than a second doseof the same attenuated RSV strain. Thus, RSV-specific immunity resultingfrom a primary immunization might be expected to restrict replication ofa second dose of an attenuated RSV strain more efficiently than that ofan HPIV-vectored virus, and indicates another potential advantage ofHPIV-vectored RSV vaccines.

The HPIV1 genome is a single strand of negative sense RNA. It consistsof a short 3′ leader region followed by 6 genes encoding the N, P, C, M,F, HN, and L proteins, and a short trailer region. Each gene encodes amajor viral protein: N, nucleoprotein; P, phosphoprotein; M, internalmatrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidaseglycoprotein; and L, major polymerase subunit. In addition, the P genecarries an overlapping ORF expressing a set of carboxy-co-terminal Caccessory proteins that inhibit the host interferon (IFN) response andblock apoptosis (Bartlett, et al. 2008. J virology 82:8965-8977). Likeother nonsegmented negative strand RNA viruses, HPIV1 transcriptioninitiates at the 3′ end promoter and proceeds down the genome in astart-stop process regulated by the gene end (GE)-intergenic (IG)-genestart (GS) signals to generate a series of monocistronic mRNAs. There isa 3′ to 5′ gradient of decreasing transcription, with thepromoter-proximal genes expressed at higher levels (Nagai. 1999. Reviewsin medical virology 9:83-99). Like other paramyxoviruses, completeinfectious, replication-competent HPIV1 can be recovered in cell culturefrom transfected cDNAs (reverse genetics).

Previous studies have described the development of a chimera of bovineand human PIV3 as a vector for RSV F protein (Schmidt et al 2000 J Virol74:8922-8929; Schmidt et al 2001 J Virol 75:4594-4603; Schmidt et al2002 J Virol 76:1088-1089; Tang et al 2002 J Virol 78:11198-11207;Bernstein et al 2012 Pediatr Infect Dis 31:109-114; see also Example 1).This virus, called rB/HPIV3, consists of BPIV3 in which the F and HNgenes were replaced using reverse genetics with those of HPIV3,combining the attenuation phenotype of BPIV3 in primates with the majorneutralization antigens of HPIV3 (Schmidt et al 2001 J Virol75:4594-4603; Schmidt et al 2002 J Virol 76:1088-1089; Tang et al 2002 JVirol 78:11198-11207; Bernstein et al 2012 Pediatr Infect Dis31:109-114) rB/HPIV3 was shown to efficiently express the RSV F and Ggenes. Clinical evaluation of a lead rB/HPIV3/RSV-F construct as abivalent vaccine for RSV and HPIV3 in seronegative children showed thatit was infectious, well tolerated, and attenuated, but was lessimmunogenic against RSV F than hoped (Bernstein et al 2012 PediatrInfect Dis 31:109-114). This appeared to be due at least in part togenetic instability in the clinical trial material that silencedexpression of the RSV F insert (Yang, et al. 2013. Vaccine31:2822-2827). However, further studies are underway to stabilize theRSV F insert and to obtain increased immunogenicity by characterizingand optimizing various parameters of vector construction (Liang, et al.2014. J virology 88:4237-4250). HPIV1 is another attractive vector forexpressing RSV F antigen. In particular, HPIV1 infects somewhat later inchildhood than RSV or HPIV3 (Counihan, et al. 2001. Pediatric infectiousdisease journal 20:646-653; Reed, et al. 1997. J Infect Dis175:807-813), and so an HPIV1-vectored RSV vaccine might be usedsubsequent to a live attenuated RSV or rB/HPIV3-vectored vaccine toboost immune responses to RSV.

In the present study, two parameters for developing an HPIV1-vectoredvaccine expressing RSV F protein were evaluated (FIGS. 36 and 42): (i)two different attenuated HPIV1 backbones were compared, and (ii)insertion of the RSV F gene at the first, second, and third genomepositions of the HPIV1 vector was compared. The first parameter, thelevel of attenuation, is important because it is linked to safety and,inversely, to immunogenicity. Specifically, the HPIV1 vector must besufficiently attenuated so as to be non-pathogenic and well tolerated,but must replicate and express antigens sufficiently well to besatisfactorily immunogenic. The addition of a foreign gene, such as RSVF, to an HPIV vector also typically confers attenuation and also canconfer the temperature-sensitivity (ts) phenotype (Liang, et al. 2014. Jvirology 88:4237-4250), and so the combined effect of the insert andspecific attenuating mutation(s) had to be determined. The two differentHPIV1 backbones used in the present study each contained a singleattenuating mutation (C^(Δ170) or L^(Y942)A) developed in previousstudies (1, 3, 25). Each of these mutations has been shown to bemoderately attenuating in vivo (Bartlett, et al. 2006. Vaccine24:2674-2684; Bartlett, et al. 2007. Virology J 4:67; Newman, et al.2004. J Virol 78:2017-2028). The C^(Δ170) mutation is non-temperaturesensitive. It reduces the ability of C proteins to inhibit the host typeI interferon response and apoptosis (Bartlett, et al. 2006. Vaccine24:2674-2684; Bartlett, et al. 2008. J virology 82:8965-8977; Newman, etal. 2004. J Virol 78:2017-2028) resulting in virus attenuation. Themechanism of attenuation has the potential to increase the inherentimmunogenicity of the construct because this mutation in C increases thehost interferon response and apoptosis response, both of which have thepotential to increase immunogenicity. The C^(Δ170) mutation consists ofa 6-nucleotide deletion in the overlapping P and C ORFs. In the C ORF,this results in the deletion of two amino acids and the substitution ofa third amino acid (specifically, the triplet 168-RDF-170 was changed tothe single amino acid S), whereas in the overlapping P ORF it results indeletion of two amino acids (172-GF-173). Deletion mutations are thoughtto have increased stability because they offer greater genotypic andphenotypic stability due to low risk of same site reversion. TheL^(Y942A) mutation is temperature-sensitive. It is a missense mutation(942-Y to A) in the L ORF that was designed to involve 3 ntsubstitutions so as to be highly resistant to de-attenuation (FIG. 42),as has been directly documented (McAuliffe et al 2004. J Virol78:2029-2036). (ii) The second parameter investigated in the presentstudy, the position of insertion of the foreign gene in the HPIV genome,is important because it affects the level of expression of the foreigngene as well as its attenuating impact on the vector. In an HPIV genome,insertion of the RSV F gene closer to the promoter would be expected toprovide a higher level of expression, due to the transcription gradient.However, the closer the foreign gene is to the promoter, the greater thenumber of downstream vector genes it can impact, because each of thesevector genes is now one position further removed from the promoter andconsequently is expressed less efficiently. In addition, placement ofthe foreign gene in the first position has the potential to affect thefunctioning of the promoter. The insertion of the foreign gene also canhave unpredictable effects. It also can be attenuating through effectssuch as the increase in genome length and gene number.

Six viruses, representing three different insertion sites for RSV F intwo different attenuated HPIV1 backbones, were constructed, rescued byreverse genetics, and analyzed for in vitro replication and expressionof RSV F and vector proteins. The hamster model was used to assess invivo replication (upper and lower respiratory tract), vaccine virusstability, immunogenicity, and protection against wt RSV challenge.

Materials and Methods

Cells and Viruses. LLC-MK2 (ATCC CCL-7) rhesus monkey kidney and Vero(ATCC CCL-81) African green monkey kidney cell lines were maintained inOpti-MEMI medium with GlutaMAX (Life Technologies, Grand Island, N.Y.)supplemented with 5% fetal bovine serum (FBS; HyClone/Logan, UT) and 1mM L-glutamine (Life Technologies). BSR T7/5 cells are baby hamsterkidney 21 (BHK-21) cells that constitutively express T7 RNA polymerase(Buchholz, et al. 1999. J Virol 73:251-259). These cells were maintainedin Glasgow minimal essential medium (GMEM; Life Technologies)supplemented with 10% FBS, 2 mM L-glutamine and 2% MEM amino acids (LifeTechnologies). Medium was also supplemented with 2% Geneticin (LifeTechnologies) at every other passage to select for cells that posses theT7 polymerase construct.

HPIV1 was propagated in LLC-MK2 cells. Before virus inoculation, LLC-MK2cells, grown in media containing 5% FBS, were washed twice with 1×phosphate buffered saline (PBS) to remove FBS. Infection with HPIV1 wasalways performed in serum-free Opti-MEMI media containing 1.2% trypsin(TrypLE Select; Life Technologies), 100 U/ml Penicillin, 100 μg/mlStreptomycin (Life Technologies) and 1 mM L-glutamine. Infected cellswere incubated at 32° C. till the appearance of cytopathic effects. Forvirus stock harvest, culture supernatant was harvested and clarified bycentrifugation at 1500 rpm for 10 min at 4° C. Aliquots of virus stockswere snap-frozen on dry ice and stored at −80° C. HPIV1 titers weredetermined by 10-fold serial dilutions in 96-well plates on LLC-MK2cells with serum-free Opti-MEMI media containing 1.2% trypsin asdescribed above followed by incubation at 32° C. for 7 days. Infectedcells were detected by hemadsorption (HAD) using guinea pig erythrocytesand titers were calculated as log₁₀ tissue culture infective dose 50%(TCID₅₀/ml) as previously described (Bartlett, et al. 2006. Vaccine24:2674-2684). The temperature sensitivity (ts) phenotype of each of thevirus was studied by evaluating their efficiency of replication at 32,35, 36, 37, 38, 39, and 40° C. as previously described Skiadopoulos, etal. 1999. Vaccine 18:503-510). Titration of each virus was performed in96-well replicate plates of LLC-MK2 cells, as described above, andincubated in sealed containers in temperature-controlled water baths atvarious temperatures for 7 days. Titers were determined by HAD andreported as TCID₅₀/ml.

Design of rHPIV1-C^(Δ170) and rHPIV1-L^(Y942A) Viruses Expressing theRSV F Antigen.

The rHPIV1 viruses were constructed using the reverse genetic systemderived from the wild type (wt) HPIV1 strain Washington/20993/1964(GenBank accession AF457102) (Newman, et al. 2002. Virus Genes24:77-92). The recombinant full-length antigenomic cDNA clone (pFLC) ofHPIV1 was modified by site-directed mutagenesis to contain 3 additionalunique restriction sites: MluI (ACGCGT, pre-N position, nucleotidenumbers 113-118), AscI (GGCGCGCC, N-P position, nucleotide numbers1776-1783) and NotI (GCGGCCGC, P-M position, nucleotide numbers3609-3616). Two attenuated cDNA backbones were generated by introducingeither the C^(A170) (Bartlett, et al. 2007. Virology J 4:67) or theL^(Y942A) (Bartlett, et al. 2007. Virology J 4:67; McAuliffe, et al.2004. J virology 78:2029-2036) mutation into the P/C or L ORF,respectively, using the QuikChange Lightning Mutagensis Kit (Agilent,Santa Clara, Calif.) as per manufacturer's instructions. The followingmutagenesis primers were used to generate the attenuated HPIV1backbones. For HPIV1 C^(Δ170) mutation, the forward primer wasAAGAAGACCAAGTTGAGCCAGAAGAGGTACGAAG (SEQ ID NO: 121) and the reverseprimer was CTTCGTACCTCTTCTGGCTCAACTTGGTCTTCTT (SEQ ID NO: 122). Theseprimers introduced a 6-nucleotide deletion (GGATTT) between positions 17and 18 compared to the P/C ORF (FIG. 42) keeping with the rule of six(Calain, et al. 1993. J Virol 67:4822-4830; Kolakofsky, et al. 1998. JVirol 72:891-899). As previously described (Bartlett, et al. 2007.Virology J 4:67), the C^(Δ170) deletion in the P/C ORF includes an R168Ssubstitution and a deletion of D and F from position 169-170 resultingin a change in the amino acid sequence of C protein from RDF to S. Thismutation also affects the P ORF involving a deletion of two residues Gand F from position 172-173. For the L^(Y942A) mutation, the forwardprimer was CCAGCTAACATAGGAGGGTTCAACGCGATGTCTACAGCTAGATGTTTTGTC (SEQ IDNO: 123) and the reverse primer wasGACAAAACATCTAGCTGTAGACATCGCGTTGAACCCTC CTATGTTAGCTGG (SEQ ID NO: 124).In these primer sequences the mutation site TAT (Y) to GCG (A) at aa 942in the L ORF is underlined. Mutagenesis primer pairs were PAGE 2-Steppurified (Operon, Huntsville, Ala.). Clones with the desired mutation,as determined by sequencing, were then purified by plasmid maxiprep(EndoFree Plasmid Maxi Kit; Qiagen) and sequenced in entirety. TherHPIV1 pFLCs with either the C^(Δ170) or the L^(Y942A) mutation weredigested with MluI, AscI, or NotI enzymes (New England Biolabs, IpswichMass.), treated with Calf Intestinal Phosphatase (New England Biolabs),and purified by gel extraction (QIAEX II Gel Extraction Kit; Qiagen,Valencia Calif.).

The RSV F gene from strain A2 with HEK amino acid assignments: Glu andPro at aa position 66 and 101, respectively (Whitehead, et al. 1998. Jvirology 72:4467-4471) was optimized for human codon usage (GeneArt,Life Technologies, Grand Island, N.Y.). RSV F gene insert was designed(FIG. 36) to include the HPIV1 transcriptional regulatory sequences:gene end (GE)-intergenic (IG)-gene start (GS) signals to allow for RSV Fexpression as an independent transcript. For all constructs, the RSV Fgene insert was designed to include HPIV1 N GE (AAGTAAGAAAAA, SEQ ID NO:125) and P GS (AGGGTGAATG, SEQ ID NO: 126) sequences along with theconserved IG sequence (CTT), while keeping the +1 phasing for the P GS.RSV F inserts were generated by PCR with the corresponding flankingMluI, AscI, or NotI restriction sites complimentary to those in theHPIV1 FLCs for insertion in the first Pre-N (F1), second N-P (F2), orthird P-M (F3) positions, respectively (FIG. 36). The following PCRprimers were used to generate the RSV F inserts. For RSV F fragmentscontaining MluI restriction sites for insertion into the first F1 geneposition the forward primer was ACGCGTCCCGGGAACAATGGAACTGCTGATCCTGAAGGCCAACGCC (SEQ ID NO: 127) and the reverse primer wasACGCGTCGTACGCATTCACCCTAAGTTTTTCTTACTTTCTATCAGTTGGAGAAGGCGATATTGTTGATGCCGG (SEQ ID NO: 128).

(SEQ ID NO: 111) For RSV F fragments containing the AscIrestriction sites for insertion into the second F2gene position the forward primer was GGCGCGCCCCCGGGAACAATGGAACTGCTGATCCTGAAGGCCAACGCC (SEQ ID NO: 129) and the reverse primer wasGGCGCGCCCGTACGCCATTCACCCTAAGTTTTTCTTACTTGATTCTATCAGTTGGAGAAGGCGATATTGTTGATGCCGG(SEQ ID NO: 130). For RSV F fragments containingthe NotI restriction sites for insertion into thethird F3 gene position the forward primer wasGCGGCCGCCCGGGAAGTAAGAAAAACTTAGGGTGAATGAACAATGGAACTGCTGATCCTGAAGGCCAACGCC(SEQ IDNO: 131) and the reverse primer was GCGGCCGCCGTACGCTATCAGTTGGAGAAGGCGATATTGTTGATGCCGG.

Recovery of rHPIV1 C^(Δ170) and rHPIV1 L^(Y942A) Viruses Expressing theRSV F Antigen.

The rHPIV1 C^(Δ170) backbones expressing RSV F from the first (HPIV1C^(Δ170)-F1), second (HPIV1 C^(Δ170)-F2), and third (HPIV1 C^(Δ170)-F3)positions and the rHPIV1 L^(Y942A) viruses expressing RSV F from thefirst (HPIV1 L^(Y942A)-F1), second (HPIV1 L^(Y942A)-F2), and third(HPIV1 L^(Y942A)-F3) positions were rescued from cDNA by using the HPIV1reverse genetics system (Newman, et al. 2002. Virus Genes 24:77-92) inBSR T7/5 cells constitutively expressing T7 RNA polymerase (Buchholz etal. 1999. J. Virology 73:251-259). This rHPIV1 cDNA is derived from theWashington/20993/1964 (HPIV1 WASH/64) clinical isolate. The BSR T7/5cells were grown to 90% confluency in 6-well plates and co-transfectedwith the antigenome pFLC plasmid (5 ug) to be rescued along with pTM-N(0.8 ug), pTM-P (0.8 ug) and pTM-L (0.1 ug) support plasmids expressingHPIV1 N, P and L proteins, respectively, using Lipofectamine 2000 (20ul) (Life Technologies) as previously described (Bartlett, et al. 2005.Vaccine 23:4631-4646; Newman, et al. 2002. Virus Genes 24:77-92).Transfected cells were incubated overnight at 37° C., washed twice withOptiMEM media (Life Technologies, Grand Island, N.Y.) and fresh OptiMEMcontaining 1 mM L-glutamine and 1.2% trypsin was added to the cellsfollowed by incubation at 32° C. At 48 h post-transfection, cells wereharvested by scraping into the medium and the cell suspension was addedto 50% confluent monolayers of LLC-MK2 cells in OptiMEM, 1 mML-glutamine, 1.2% trypsin (Life Technologies) and incubated at 32° C.Virus was harvested after 7 days and was further amplified by one(HPIV1-C^(Δ170)) or two (HPIV1-L^(Y942)A) passages in LLC-MK2 cells at32° C. Virus titers were determined by 10-fold serial dilutions onLLC-MK2 cells in 96-well plates as described above. All recombinantviruses were sequenced to confirm the lack of adventitious mutations.For this, viral RNA was extracted (QiaAmp Viral RNA Mini Kit; Qiagen,Valencia, Calif.) from virus stocks and treated with RNase free DNase I(Qiagen) to remove the plasmid DNA used for virus rescue. RNA wasreverse transcribed (SuperScript First-Strand Synthesis System forRT-PCR; Invitrogen/Life Technologies) and overlapping genome regionswere amplified by RT-PCR (Advantage-HF 2 PCR Kit; ClontechLaboratories). RT-PCR controls lacking the reverse transcriptase wereincluded for all viruses, which showed that the amplified products werederived from viral RNA and not from the pFLC cDNA used for virusrecovery. The genome sequence of each virus construct was determined bydirect Sanger sequencing of the overlapping amplified RT-PCR products.Sequence reads were aligned and the genome sequence was assembled usingSequencher program-version 5.1 (Gene Codes Corporation, Ann Arbor,Mich.).

Replication of Chimeric rHPIV1 Viruses in Vero and LLC-MK2 Cells.

Triplicate wells of Vero or LLC-MK2 cell monolayers in 6-well plateswere infected at a multiplicity of infection (MOI) of 0.01 TCID₅₀ withHPIV1 (C^(Δ170) or L^(Y942A)) viruses expressing RSV F (F1, F2, or F3),or the empty HPIV1 C^(Δ170) or HPIV1 L^(Y942A) vector, or wt HPIV1.Cultures were incubated at 32° C. Aliquots of 0.5 ml from a total 2 mlcell culture supernatant medium were collected at 24 h intervals fromeach well and replaced by fresh media. The samples were flash frozen andstored at −80° C. Virus titers (log₁₀ TCID₅₀/ml) were determined byserial dilution on LLC-MK2 cells followed by detection of infected cellsby HAD as described above.

Analysis of RSV F and HPIV1 Vector Protein Expression by WesternBlotting.

Vero cells (1×10⁶) were infected with HPIV1 C^(Δ170) or HPIV1 L^(Y942A)constructs expressing RSV F from either of the three genome positions(F1, F2, or F3). Empty HPIV1 C^(Δ170) and HPIV1 L^(Y942A) vector, wtHPIV1, and mock treated cells were used as controls. Infections wereperformed at an MOI of 5 TCID₅₀ per cell and incubated at 32° C. At 48 hpost-infection (post-infection), monolayers were washed twice with PBSand lysed with 400 ul of 1×LDS sample buffer (Life Technologies). Forelectrophoresis, lysates were reduced and denatured by mixing with 1×reducing reagent (Life Technologies) and incubation at 37° C. for 30min. Reduced denatured lysate (40 μl) was loaded onto 4 to 12% Bis-TrisNuPAGE gels (Novex-Life Technologies) and electrophoresis performed in1×MOPS buffer. Proteins were transferred onto PVDF membranes using theiBlot protein transfer system (Life Technologies). Membranes wereblocked for 1 h in Licor blocking buffer (Licor Inc. Lincoln, Nebr.) andprobed with a murine monoclonal RSV F specific antibody (ab43812; Abcam,Cambridge, Mass.) and a rabbit polyclonal HPIV1 N specific antibody(HPIV1-N-485) at 1:1000 dilution in blocking buffer. HPIV1-N-485 wasgenerated by immunization of rabbits with the KLH-conjugated N peptidespanning the amino acid (aa) residues 485-499 of N as previouslydescribed (Bartlett, et al. 2010. Vaccine 28:767-779). Replicate blotsperformed with the same set of lysates were probed with the rabbitpolyclonal antisera for HPIV1 P (SKIA-1), F (SKIA-15), or HN (SKIA-13)which were also raised by repeated immunization of rabbits with theKLH-conjugated peptide, and were used at 1:200 dilution. After overnightincubation with the above antibodies, the membranes were washed 4×, 5min each, followed by incubation with the secondary antibodies, dilutedin the Licor blocking buffer, for 1 h. The corresponding infra-reddye-conjugated secondary antibodies were goat anti-mouse IRDye 680LT andgoat anti-rabbit IRDye 800CW (LiCor). Membranes were scanned and theblot images were acquired using an Odyssey infrared imaging system(LiCor). Fluorescence intensities of the protein bands, derived fromthree independent experiments, were quantified by using the Licor imageanalysis suite (Image Studio) and reported as expression of RSV F orHPIV1 vector proteins (N, P, F, and HN) relative to F3 viruses.

Percentage of Virions Expressing RSV F Determined by FluorescentDouble-Staining Plaque Assay.

An infrared fluorescence based two-color plaque assay was developed tosimultaneously detect the expression of RSV F and HPIV1 proteins in theplaques formed by the HPIV1 vectors expressing RSV F on Vero cellmonolayers. Each virus was 10-fold serially diluted in OptiMEM mediacontaining 1 mM L-glutamine and 1.2% trypsin and 100 μl of each dilutionwas added in duplicate to Vero cell monolayers grown in 24-well plates.Inoculated cells were incubated for 2 h at 32 C on a rocker after whichan overlay OptiMEMI media containing 0.8% methylcellulose (SigmaAldrich, St. Louis, Mo.), 1 mM L-glutamine, 4% trypsin, 100 U/mlPenicillin, and 100 μg/ml Streptomycin was added to each well. Foranimal tissue derived virus samples, Timentin (200 mg/ml), Ampicillin(100 mg/ml), Cleocin (150 mg/ml), and Amphotericin B (250 μg/ml) wereincluded in the methylcellulose overlay instead of Penicillin andStreptomycin. After incubation for 6 days at 32 C, cells were fixedtwice with 80% cold methanol. The rHPIV1-RSV F plaques were detected oninfected Vero cell monolayers by co-immunostaining with a mixture ofthree RSV F-specific monoclonal antibodies at a 1:2000 dilution each, aspreviously described (Murphy, et al. 1990. Vaccine 8:497-502) and anHPIV1 specific goat polyclonal antibody (ab20791; Abcam) at a 1:1600dilution in Licor blocking buffer. After 1 h incubation, cells werewashed once with 1 ml blocking buffer and incubated with the secondaryantibody mixture containing infrared dye-conjugated goat anti-mouse680LT and the donkey anti-goat 800CW (LiCor) each at a 1:800 dilution inthe blocking buffer. Cells were washed twice with 1×PBS and images ofthe co-stained plaques were acquired by scanning the plates on anOdyssey infrared imaging system (LiCor). Wells containing fewer than 50plaques were chosen for analysis and the percentage of rHPIV1 plaquespositive for RSV F expression was determined. This assay was employed toassess the stability of RSV F expression by determining the percentpopulation expressing RSV F in the vaccine inoculum as well as in thevirus isolated from hamsters after in vivo replication.

Hamster Studies

Virus Replication in Hamsters.

All animal studies were approved by the National Institutes of Health(NIH) Institutional Animal Care and Use Committee (IACUC). In vivoreplication of each virus at 3 and 5 days post-infection as well as theimmunogenicity was assessed in hamsters. Six-week old Golden Syrianhamsters were confirmed to be seronegative for HPIV1 and RSV byanalyzing pre-immune sera by hemagglutination inhibition (HAI) assay andan RSV neutralization assay, respectively (Coates, et al. 1966. Am JEpidemiol 83:299-313; Coates, et al. 1966. J Bacteriol 91:1263-1269; vanWyke Coelingh, et al. 1988. J Infect Dis 157:655-662). Groups of 6hamsters per virus were anesthetized and inoculated intranasally with0.1 ml L15 medium (Life Technologies) containing 10⁵ TCID₅₀ of the virusper animal. To evaluate replication, hamsters were euthanized on days 3and 5 post-infection and nasal turbinates and lungs were collected forvirus titration. Tissue homogenates in L15 medium containing Timentin(200 mg/ml), Ampicillin (100 mg/ml), Cleocin (150 mg/ml), andAmphotericin B (250 ug/ml) were titrated by serial dilution on LLC-MK2cells followed by detection of virus infection by HAD. Virus titers werereported as TCID₅₀/gram of hamster tissue. Tissue homogenates were alsotitrated by plaque assay on Vero cells and stained for the expression ofRSV F and HPIV1 proteins by using the fluorescent double staining plaqueassay as described above. Results were reported as percent HPIV1 plaquesexpressing RSV F.

Immunogenicity

Induction of Virus Neutralizing Antibodies (NAbs) in Serum Against RSVand HPIV1 [60% Plaque Reduction Neutralization Test (PRNT₆₀)].

To assess immunogenicity of the vaccine candidates, hamsters wereimmunized as described above and sera were collected from hamsters atday 28 after immunization. Titers of RSV specific neutralizingantibodies (NAbs) were determined by PRNT₆₀ on Vero cells as previouslydescribed (Coates, et al. 1966. Am J Epidemiol 83:299-313) using theeGFP-expressing RSV (Munir, et al. 2008. J Virol 82:8780-8796). Hamstersera were incubated at 56° C. for 30 min to inactivate the complementproteins followed by serial dilution in Opti-MEMI, 2% FBS, 1× Gentamicinmedia in 96-well plates. RSV-eGFP diluted in Opti-MEM, 2% FBS, 1×Gentamicin, 10% guinea pig complement (Yoder, et al. 2004. J medicalvirology 72:688-694) (Lonza, Walkersville, Md.) was further diluted 1:1by mixing with an equal volume of serially diluted serum samplesfollowed by 30 min incubation at 37° C. A volume of 100 μl ofserum-virus mix was added to Vero cells grown in 24-well plates andvirus was allowed to adsorb for 2 h on the rocker at 32° C. An overlayof Opti-MEMI, 8% methylcellulose, 2% FBS, 1× Gentamicin was added andincubated for 5-6 days to allow plaque formation. RSV plaques onmonolayers were visualized by scanning and acquiring images usingTyphoon Imager (GE Healthcare, Piscataway, N.J.). Plaque counts for eachsample were determined and the NAb titer was determined as described(Coates, et al. 1966. Am J Epidemiol 83:299-313). Titers ofHPIV1-specific NAbs were also determined by 60% plaque reduction assayon Vero cells, using the methods as described for RSV, usingGFP-expressing rHPIV1 with the following modifications: First, in caseof HPIV1 neutralization assay, guinea pig complement was not used as itwas found to neutralize the virus, second, the inoculated Vero cellswere washed twice with 1×PBS after virus adsorption to remove serum, andthird a methylcellulose overlay medium containing 4% trypsin and lackingFBS was used.

The ability of vaccine candidates to protect against RSV infection wastested by challenge infection of hamsters at 30 days post-immunizationby intranasal inoculation with 0.1 ml L15 medium containing 10⁶ PFU ofwt RSV strain A2. Hamsters were euthanized and nasal turbinates andlungs were harvested 3 days post-challenge and viral loads of challengeRSV in these tissues were determined by plaque assay on Vero cells(Durbin, et al. 2003. Clinical infectious diseases 37:1668-1677; Luongo,et al. 2013. J virology 87:1985-1996).

Results

Creation of Two Attenuated HPIV1 Backbones (rHPIV1 C^(Δ170) and rHPIV1L^(Y942A)) Expressing the RSV F Protein from Three Different GenomeLocations.

Two attenuated HPIV1 backbones were prepared that each contained adifferent, previously identified attenuating mutation (namely, C^(Δ170)and L^(Y942)A)) that had been designed for stability againstde-attenuation (Introduction, FIGS. 36 and 42). The C^(Δ170) mutationdid not confer temperature-sensitivity while the L^(Y942A) mutation wasa temperature sensitivity mutation. Next, the RSV F ORF of strain A2 wascodon-optimized for human expression and engineered to be under thecontrol of HPIV1 gene-start and gene-end signals, and was inserted intothe rHPIV1 C^(Δ170) and rHPIV1 L^(Y942A) backbones at three different,parallel genome locations: namely at the first gene position (Pre-N,yielding rHPIV1 C^(Δ170)-F1 and rHPIV1 L^(Y942A)-F1); at the second geneposition (N-P, yielding rHPIV1 C^(Δ170)-F2 and rHPIV1 L^(Y942A)-F2): andat the third gene position (P-M, yielding rHPIV1 C^(Δ170)-F3 and rHPIV1L^(Y942A)-F3) (FIG. 36). Each construct conformed to the “rule of six”(Calain, et al. 1993. J Virol 67:4822-4830). Each vector gene maintainedits original hexamer spacing, while the F1, F2, and F3 inserts assumedthe original hexamer spacing of the N, P, and P genes, respectively. TheRSV F protein also carried the HEK amino acid assignments, Glu and Proat residues 66 and 101, respectively (Whitehead, et al. 1998. J virology72:4467-4471) which matches with the sequence of RSV F from earlypassages of strain A2 and also is consistent with most other clinicalisolates.

The rHPIV1/RSV F viruses were recovered by reverse genetics. All viruseswere rescued readily except for the rHPIV1 L^(Y942A)-F2 construct, whichappeared to be recovered with low efficiency and required multiplepassages to make a working pool. The complete sequence of each virus wasdetermined to verify the absence of adventitious mutations. All of therHPIV1/RSV-F virus working pools were free of apparent adventitiousmutations except for the rHPIV1 L^(Y942A)-F2 virus: for this virus, nineclones were rescued of which only one had the correct genome sequencelacking adventitious mutations. The other eight clones containedadventitious mutations, which were predominantly in the HPIV1transcriptional signals downstream of the HPIV1 N gene (N geneend-intergenic-P gene start) and preceding the RSV F ORF. Since theinsertion sites of the RSV F gene in the second positions of theparallel rHPIV1 L^(Y942A)-F2 and rHPIV1 C^(Δ170)-F2 constructs wereidentical, this suggests that the problem with the former virus wasspecific to the L^(Y942A) mutation (e.g., altered polymerase function)rather than the insertion site by itself.

Replication of HPIV1/RSV F Viruses in Vero and LLC-MK2 Cells.

Replication of HPIV1/RSV viruses was evaluated in vitro by determiningtheir multistep growth kinetics in Vero (FIGS. 37A and 37C) and LLC-MK2(FIGS. 37B and 37D) cells. Cells were infected at an MOI of 0.01 TCID₅₀and incubated at 32° C. Supernatant was harvested at 24 h intervals over7 days and virus titers were determined by HAD and reported as TCID₅₀/ml(FIG. 37). The Student t test was used to determine the statisticalsignificance of difference between the titer of each virus versus wtHPIV1 for day 2 and 7 p.i. On day 7, all viruses replicated to finaltiters greater than 7.2 log₁₀ TCID₅₀/ml in Vero cells and 7.4 log₁₀TCID₅₀/ml in LLC-MK2 cells, with slight differences among the virusesthat were statistically insignificant compared to wt HPIV1 in both celllines (FIG. 37). However, differences were observed at earlier timepoints, especially day 2 p.i. In the case of the rHPIV1 C^(Δ170) viruses(FIGS. 37A and B), the rHPIV1 C^(Δ170) empty vector and rHPIV1C^(Δ170)-F3 replicated similar to wt HPIV1 in both cell lines on day 2post-infection. However, the replication of rHPIV1 C^(Δ170)-F1 wassignificantly reduced in Vero (p<0.001) and LLC-MK2 (p<0.05) cells andthat of the rHPIV1C^(Δ170)-F2 was significantly reduced (p<0.01) in Verocells. For the rHPIV1 L^(Y942A) viruses (FIGS. 37C and 37D), replicationof the rHPIV1 L^(Y942)A empty vector was significantly lower than thatof the wt rHPIV1 in Vero cells, but both grew to similar titers inLLC-MK2 cells on day 2 post-infection. Highly significant reductions(p<0.0001) in replication as compared to wt rHPIV1 were observed forrHPIV1 L^(Y942A)-F1, -F2 and -F3 viruses in Vero cells. Likewise, therHPIV1 L^(Y942A)-F1, -F2, and -F3 viruses showed significantly reduced(p<0.01, p<0.0001, and p<0.01, respectively) replication in LLC-MK2cells. In Vero cells, rHPIV1 L^(Y942A)-F1 grew at the same rate as itsparent rHPIV1^(LY942A) empty vector while the growth of rHPIV1L^(Y942A)-F2 and -F3 viruses (FIG. 37C) was relatively reduced, butthese differences among the chimeric viruses were statisticallyinsignificant.

Expression of RSV F and HPIV Vector Proteins by the ChimericrHPIV1/RSV-F Viruses.

Expression of the RSV F protein and the HPIV1 vector N, P, F and HNproteins was evaluated for all viruses by Western blot analyses. Verocells were infected at an MOI of 5 TCID₅₀ and incubated for 48 h at 32°C. Denatured and reduced lysates were subjected to SDS-PAGE and Westernblot, and were analyzed using antibodies specific to each individualprotein. RSV F is initially translated as the F0 precursor that ispost-translationally cleaved by furin-like protease intodisulfide-linked F1 and F2 subunits. Immunostaining with a monoclonalantibody specific to RSV F detected both F0 (70 kD) and F1 (48 kD). TheHPIV1 N, P, F, and HN proteins were detected with correspondinganti-peptide polyclonal antibodies. Representative blots from one ofthree independent experiments are shown in FIG. 38A, and results fromall three experiments are quantified for the rHPIV1 C^(Δ170) and rHPIV1L^(Y942A) constructs in FIGS. 38B and C, respectively, with the valuesnormalized relative to those of the F3 construct in each series as 1.0.

The HPIV1 C^(Δ170)-F1, -F2, and F3 viruses expressed substantial amountsof RSV F (FIG. 38A, lanes 1-3, and FIG. 38B) that were only slightlyhigher for F1 and F2 as compared to F3 as indicated by the protein bandquantification (FIG. 38B) with the differences being statisticallyinsignificant. Thus, unexpectedly, a 3′-5′ polar gradient of expressionfrom F1 to F3 was not observed. In contrast, however, a strong polargradient of RSV F expression was observed in case of HPIV1 L^(Y942A)viruses, with a significantly higher expression of RSV F by the F1 virusas compared to the F2 (p<0.01) and F3 (p<0.05) viruses (FIG. 38A, lanes5-6, and FIG. 38C).

The expression of the vector N, P, F, and HN proteins was evaluated forwt HPIV1, the empty vectors, and the F1, F2, and F3 constructs in theC^(Δ170) and L^(Y942A) series. In general, the C^(Δ170) mutation did notaffect vector protein expression, with the result that the rHPIV1C^(Δ170) empty vector had a vector protein expression profile similar tothat of wt rHPIV1 (FIG. 38B). In the case of the three versions ofrHPIV1C^(Δ170) vector expressing RSV F, the F3 virus expressed N, P, F,and HN at levels similar to that of empty rHPIV1C^(Δ170) vector. The F2virus expressed N protein similar to the empty rHPIV1C^(Δ170) vector butshowed reduced expression of P, F, and HN proteins of which only the HNreduction was statistically significant compared to the empty vector.The F1 virus demonstrated significant reduction in all vector proteinsincluding N (p<0.05), P (p<0.01), F (p<0.05), and HN (p<0.05) comparedto the empty vector. Thus, insertion of the RSV F gene into the rHPIV1C^(Δ170) vector reduced the expression of downstream vector genes exceptin the F3 virus. Both the F1 and F2 viruses exhibited significantlyreduced replication early during infection of Vero cells (FIG. 37A), andthe reduced synthesis of vector proteins provides a plausibleexplanation.

In contrast to the C^(Δ170) mutation, the L^(Y942A) mutation did affectthe expression of vector proteins: specifically, compared with wt HPIV1,the rHPIV1 L^(Y942A) empty vector had significantly reduced expressionof the P (p<0.05), F (p<0.05), and HN (P<0.01) proteins, with nosignificant difference for the N protein (FIGS. 38A, C). In case of thethree versions of rHPIV1 L^(Y942A) expressing RSV F, the F3 virus hadmodest reductions in the abundance of vector proteins as compared withthe empty backbone, but none of the reductions was significant.Interestingly, this lack of significant vector protein reduction wasconsistent with the rHPIV1 C^(Δ170)-F3 virus, suggesting that the F3insert position does not interfere with vector protein expression andprovides a site for which interference with the vector is minimal. TheF2 virus on the other hand exhibited highly pronounced and significantreduction in N, P, F, and HN proteins as compared to the rHPIV1L^(Y942A) empty vector. In comparison, the rHPIV1 C^(Δ170)-F2 virus,also showed reduced expression of the P, F, and HN proteins but not theN protein. This was to be expected because insertion after N at thesecond F2 position might be expected to reduce the expression ofdownstream genes due to the 3′-5′ polar transcriptional gradient,whereas the upstream N gene might be expected to be unaffected, as wasobserved. Unexpectedly, however, in case of the rHPIV1 L^(Y942A)-F2,although RSV F was inserted downstream of the N gene, the expression ofN protein was also negatively affected. The rHPIV1 L^(Y942A)-F1 hadsignificantly reduced expression only in the case of the N protein,while expression of the P, F, and HN proteins was comparable to that ofthe rHPIV1 L^(Y942A) empty vector.

Comparison of Syncytium Formation by rHPIV1/RSV-F Vectors.

A hallmark of RSV infection in vitro is the characteristic syncytiumformation mediated by the RSV F protein. Infection of LLC-MK2 cellmonolayers by wt HPIV3 or the rHPIV1 C^(Δ170) or L^(Y942A) empty vectorsdid not induce evident syncytium formation (FIG. 39, panels I, D, or H,respectively). In sharp contrast, HPIV1 vectors expressing high levelsof RSV F protein, namely the rHPIV1 C^(Δ170)-F1 and -F2 viruses (FIGS.39A and 39B) and the rHPIV1-L^(Y942A)-F1 virus (FIG. 39E), induced highlevels of syncytia. Syncytium formation was not evident with the rHPIV1C^(Δ170)-F3 virus (FIG. 39C) even though the level of expression of RSVF protein was only modestly less than with the F1 and F2 viruses (FIG.38). Syncytium formation also was not evident with therHPIV1-L^(Y942A)-F2 and -F3 viruses (FIGS. 39F and 39G), which expressedvery low levels of RSV F protein (FIG. 38). This functional assay(syncytium formation) suggested that the RSV F protein expressed fromthe HPIV1 vectors was folded and processed correctly and accumulated atthe cell surface in active form.

In addition, the C^(Δ170) mutation was associated with a secondcytopathic effect, namely increased apoptosis. This was most evidentwith the rHPIV1-C^(Δ170) empty vector (FIG. 39D), because the enhancedapoptosis was more readily observed in the absence of syncytiumformation. Enhanced induction of apoptosis due to the C^(Δ170) mutationmay explain why the rHPIV1-L^(Y942A)-F3 virus was inefficient ininducing syncytium formation (FIG. 39C) despite the expression of a highlevel of RSV-F (FIG. 38B): it is reasonable to suggest that the cellrounding associated with the C^(Δ170)-mediated enhanced apoptosis (e.g.,FIG. 39D) might reduce the cell-to-cell contact necessary for syncytiumformation, but very efficient expression of RSV F with other constructsmight allow syncytium formation to begin sufficiently early to overcomethe apoptosis effect (e.g., FIG. 39B versus FIG. 39C).

The observations made with LLC-MK2 cells in FIG. 39 also were obtainedwith Vero cells.

Temperature Sensitivity of the rHPIV1/RSV-F Viruses.

As noted, the C^(Δ170) mutation did not confer the ts phenotype inprevious studies, whereas the L^(Y942A) mutation did so (McAuliffe, etal. 2004. J virology 78:2029-2036; Newman, et al. 2004. J Virol78:2017-2028). Insertion of RSV F into an HPIV vector also has beenshown to confer the ts phenotype (Liang, et al. 2014. J virology88:4237-4250). The HPIV1/RSV-F constructs were therefore evaluated forthe presence and magnitude of the ts phenotype. Specifically, 10-foldserial dilutions were prepared and used to infect LLC-MK2 cells andincubated at 32, 35, 36, 37, 38, 39, and 40° C., in 7 replicates. Virustiters were determined by HAD using guinea pig erythrocytes, and titerswere reported as log₁₀ TCID₅₀/ml (FIG. 43). The reduction in titer(log₁₀) at each restrictive temperature compared to the titer at thepermissive temperature of 32° C. was calculated. The shut-offtemperature for a given virus was defined as the lowest restrictivetemperature at which the mean log₁₀ reduction in virus titer at thattemperature versus 32° C. was ≥2.0 log₁₀ compared to that of wt rHPIV1at the same two temperatures (Bartlett, et al. 2005. Vaccine23:4631-4646). The ts phenotype was defined as having a shut-offtemperature of ≤40° C.

While the C^(Δ170) mutation did not confer the ts phenotype in previousstudies, in the present study the rHPIV1 C^(Δ170) empty vector had ashut-off temperature of 40° C. (FIG. 39) and thus was ts compared to wtHPIV1, but the effect was small. The rHPIV1 C^(Δ170)-F2 and -F3constructs also had shut-off temperatures of 40° C. and thus did notdiffer significantly from the empty vector. However, the rHPIV1C^(Δ170)-F1 virus had a lower shut-off temperature of 39° C. (FIG. 43).In the present study the rHPIV1 L^(Y942A) empty vector had a shut-offtemperature of 36° C., similar to the values of 35-37° C. associatedwith this mutation in previous studies (McAuliffe et al JVI78:2029-2036, 2004; Bartlett et al Virol J 4:67, 2007). The rHPIV1L^(Y942A)-F1 and -F2 constructs were more ts than the empty vector, withshut-off temperatures of 35° C., while the rHPIV1 L^(Y942A)-F3 vectorhad the same 36° C. shut-off temperature as the empty vector (FIG. 43).Thus, insertion of the RSV F gene into the F1 position of eitherattenuated backbone increased the ts phenotype, insertion into the F3position of either backbone did not increase the ts phenotype, andinsertion into the F2 position increased the phenotype only for theL^(Y942A) backbone.

Percentage of Virions in the Vaccine Inoculum that Express RSV-F(Vaccine Stability In Vitro).

The working pools of the rHPIV1/RSV-F constructs were evaluated for thefrequency of RSV F expression in individual viral plaques using afluorescent double-staining plaque assay. Vero cells were inoculatedwith 10-fold serially diluted viruses and allowed to form plaques for 6days under a methycellulose overlay at the permissive temperature of 32°C. Viral plaques were co-immunstained for RSV F and HPIV1 proteins byusing a mix of three RSV F-specific murine monoclonal antibodies and agoat HPIV1-specific polyclonal antiserum (see Materials and Methods).The primary antibodies were detected using anti-mouse-IgG andanti-goat-IgG second antibodies conjugated with red and green infrareddyes, and the percentage of HPIV1 plaques expressing RSV F wasdetermined. A total number of 140, 77, and 59 plaques were counted forrHPIV1 C^(Δ170)-F1, -F2, and -F3, respectively, and all were found tohave 100% of the HPIV1 plaques expressing RSV F protein. F1 made plaquesof smaller size than F2 and F3 while F2 and F3 plaque size was similarto each other. A total of 214, 70, and 192 plaques were counted forrHPIV1 L^(Y942A)-F1, -F2, and -F3, respectively, that showed 100%, 100%,and 97% of the HPIV1 plaques expressing RSV F antigen. Overall, therHPIV1 L^(Y942A) viruses formed plaques of much smaller size as comparedto the rHPIV1 C^(Δ170) viruses. Since the assay was performed at thepermissive temperature (32° C.), the smaller plaque phenotype,suggesting a relatively more attenuated phenotype and slower spread, maynot be a ts effect. This was also consistent with their relativelyslower replication profile (FIG. 37).

Replication of the rHPIV1/RSV-F Viruses in the Respiratory Tract ofHamsters.

Viruses were evaluated for their ability to replicate in the upper andlower respiratory tract (URT and LRT, respectively) of hamsters.Hamsters were inoculated intranasally with a dose of 10⁵ TCID₅₀/0.1 mlper animal. To assess virus replication, hamsters were euthanized ondays 3 and 5 post-infection (6 animals per virus per day) and the nasalturbinates (URT) and lungs (LRT) were collected. Homogenates ofindividual nasal turbinates and lungs were analyzed for virusreplication by titration on LLC-MK2 cells using HAD assay, and thetiters were reported as log₁₀ TCID₅₀/ml. Virus titers are shown for days3 and 5 as open triangles and filled circles, respectively, in the URT(FIG. 40A) and the LRT (FIG. 40B). Each symbol represents an individualanimal.

Overall, the peak virus titers for all rHPIV1 vectors expressing RSV Fwere lower than that of the wt HPIV1 suggesting the attenuating effectsof the backbone mutations (C^(Δ170) or the L^(Y942A)) as well as that ofthe inserted RSV F gene. In the URT, the rHPIV1 C^(Δ170) empty vectorwas significantly attenuated on day 5 and not on day 3 but wassignificantly restricted on both days in the lungs as compared to the wtHPIV1. In the URT, significantly reduced replication was observed forrHPIV1 C^(Δ170)-F1 on day 3 (p<0.05) only and for -F2 on both days(p<0.0001); -F3 replicated at levels similar to those of wt rHPIV1 onboth days. rHPIV1 C^(Δ170)-F1, -F2, and -F3 were all significantly(p<0.0001) attenuated in the lungs on day 3 and 5; F1 and F3 wereundetectable in all and F2 was undetectable in 5 of 6 animals. TherHPIV1 L^(Y942)A empty vector replication was significantly reduced onday 3 and no virus was detected on day 5 in the URT whereas no virus wasdetected on both days in the lungs suggesting significant attenuationconsistent with the is phenotype of the L^(Y942A) mutation (McAuliffe,et al. 2004. J virology 78:2029-2036) (FIG. 43). The rHPIV1L^(Y942A)-F1, -F2, and -F3 viruses were also significantly (p<0.0001)attenuated both in the URT and lungs on day 3 and 5 with virusreplication undetectable for majority of the animals.

The HPIV1 vaccine candidate (rHPIV1-C^(R84G/Δ170)HN^(553A)L^(Y942A)),previously demonstrated to be strongly attenuated in AGMs (Bartlett, etal. 2007. Virology J 4:67) and over attenuated in sero-negativechildren, was included as a control for replication comparison and toassess the level of attenuation of the vaccine candidates beingdeveloped. As expected, this virus was significantly attenuated inhamsters with no replication observed in the URT and lungs on day 3 and5. The observation that most of the constructs developed in the presentstudy had attenuated but detectable replication suggests that they aresomewhat less attenuated than the previous vaccine candidate, which wasover-attenuated in children, suggesting that the constructs developed inthe present study may have suitable attenuation phenotypes. The chimericbovine/human PIV3 expressing RSV F from the second genome position(rB/HPIV3-F2) is being developed as a live RSV vaccine (Introduction,and Example 1). In clinical trials, this vaccine candidate was found tobe safe in 6-24 month old RSV seronegative children with a safe andwell-tolerated level of replication (Bernstein, et al. 2012. Pediatricinfectious disease journal 31:109-114). The rB/HPIV3-F2 virus wasincluded as a reference virus for the comparative assessment ofattenuation and immunogenicity of the rHPIV1 vaccine candidates beingtested. The replication level of the rHPIV1 C^(Δ170)-F1, -F2, or -F3 onboth days was either statistically similar to or significantly lowerthan that of the rB/HPIV3-F2 in both the URT and lungs. Likewise, therHPIV1 L^(Y942A)-F1, -F2, and -F3 viruses also showed significantlyreduced replication in both the URT and lungs on day 3 and 5 as comparedto rB/HPIV3-F2. These data suggest that the HPIV1 vectors with eitherthe C^(Δ170) or the L^(Y942A) backbone mutations are sufficientlyattenuated in vivo and show replication phenotype that is similar to oreven more attenuated than the rB/HPIV3-F2 virus.

In the URT, the titers for all viruses (except rHPIV1 L^(Y942A)-F1 and-F3, which did not replicate) were higher on day 3 (FIG. 40A) than onday 5. In contrast, rB/HPIV3-F2 attained peak titer on day 5. Overall,in the lungs, all vectors expressing RSV F were significantly (p≤0.0001)restricted as compared to wt HPIV1 on days 3 and 5 post-infection (FIG.40B) and were also significantly restricted as compared to rB/HPIV3-F2on day 5.

Stability of RSV F Protein Expression by the Chimeric rHPIV1 Virusesafter In Vivo Replication.

Positive selection of viruses that silence the expression of RSV F mayhappen during replication in vivo that may compromise RSV Fimmunogenicity (Yang, et al. 2013. Vaccine 31:2822-2827). Mutations mayoccur in the RSV F ORF or the regulatory transcriptional signalscontrolling its expression such that the RSV F protein expression iseither reduced or ablated. To evaluate the rHPIV1 vectors for in vivostability, Vero cells were infected with serially diluted homogenates ofthe nasal turbinates and lungs of the infected hamsters. A fluorescentdouble immunostaining plaque assay was performed to determine thepercentage of virus particles expressing RSV F. Consistent with the lackof replication in the lungs (FIG. 40B), no plaques could be detected forall vectors expressing RSV F in the lung homogenates. Similarly, noplaques were detectable for the rHPIV1 L^(Y942A) viruses expressing RSVF in the URT due to poor replication. The stability results for rHPIV1C^(Δ170) viruses, which replicated efficiently in the URT are shown(FIG. 44). These viruses were stable after in vivo replication with anoverall >98% plaques expressing RSV F on days 3 and 5 post-infection Ofthe 30 samples analyzed, 29 had 100% and only one sample had 98% (2%loss) PFUs expressing RSV F. These data suggest that the rHPIV1 C^(Δ170)vectors expressing RSV F are quite stable in hamster model and did notshow evidence of the selection of mutants with silenced RSV F expressionafter in vivo replication for at least 5 days.

Immunogenicity

Immunization with the Chimeric rHPIV1 Viruses Expressing RSV F InducesSerum Virus Neutralizing Antibodies (NAbs) Against RSV and HPIV1.

To determine the immunogenicity of the rHPIV1 viruses expressing RSV F,groups of six hamsters per virus were inoculated intranasally with 10⁵TCID₅₀ per animal. wt RSV, rHPIV1 C^(Δ170), rHPIV1 L^(Y942A) andrB/HPIV3-F2 were included as controls. Sera from immunized hamsters werecollected at 28 days post-infection and the NAb titers against RSV andHPIV1 were determined by PRNT₆₀ assay (FIG. 45). The rHPIV1 C^(Δ170)-F1,-F2, and -F3 induced RSV-specific NAbs at titers that were notstatistically different from each other. This was not surprising becauseall three viruses showed similar level of RSV F protein expression(FIGS. 38A and 38B). The rHPIV1 C^(Δ170)-F1 and -F2 had a slightlyhigher RSV F expression than the -F3 virus but this difference was notstatistically significant (FIG. 38B). Likewise, the -F1 and -F2 virusesalso showed relatively higher fusion activity and syncytia formationthan the -F3 virus that is presumably correlated with the amount of RSVF synthesized during infection (FIG. 39A-39C) Although statisticallyinsignificant, the NAb titer seems to be slightly higher for F1 and F3viruses as compared to F2 which is more consistent with their in vivoreplication profile (FIG. 40A) rather than the amount of RSV Fexpressed. The RSV NAb titer induced by rHPIV1 C^(Δ170)-F1, -F2, and -F3were significantly lower than that induced by rB/HPIV3-F2, a differencethat likely stems from the significantly reduced replication of theseviruses as compared to rB/HPIV3-F2 (FIGS. 40A and 40B).

The rHPIV1 L^(Y942A)-F1, -F2, and -F3 viruses failed to induce NAbresponse to RSV. This was unexpected because the rHPIV1 L^(Y942A)-F1showed RSV F expression similar to that of rHPIV1 C^(Δ170)-F1, -F2, and-F3 on in vitro infection of Vero cells (FIG. 38A). However, it isimportant to note that the L^(Y942A) mutation is highly attenuating andconfers a ts phenotype with a shut-off temperature of 36° C. (FIG. 43).The L^(Y942A) mutant viruses were expected to show minimal or nodetectable replication in hamsters (body temperature: 36.7 to 38.3° C.;The Merck Manual). Consistent with the backbone phenotype, the rHPIV1L^(Y942A)-F1, -F2, and -F3 were also ts with a shut off temperature of35° C., 35° C., and 36° C., respectively, indicating that the insertionof RSV F at the F1 and F2 positions reduced the shut off temperature by1° C. making them even more ts. Therefore, although the rHPIV1L^(Y942A)-F1 was able to express RSV F at levels comparable to those ofthe rHPIV1 C^(Δ170)-F1, -F2, and -F3 viruses at permissive temperature(FIG. 38A), it failed to induce RSV NAbs likely due to its strong tsphenotype and inability to replicate in vivo. The rHPIV1 L^(Y942A)-F2and -F3 viruses seemingly had two confounding factors: they showedrelatively poor RSV F expression (FIGS. 38A and 38C) and in additionwere ts and did not replicate in vivo (FIGS. 40A and 40B), resulting inlack of an RSV NAb response. Thus, the rHPIV1 L^(Y942A)-F1, -F2, and -F3viruses were not immunogenic and did not generate vaccine candidatesbecause of their over attenuated ts phenotype and lack of replicationand immunogenicity in vivo.

The HPIV1 specific NAb response in hamsters was also evaluated by PRNT₆₀assay (FIG. 45). Overall, the levels of HPIV1 NAb titers were lower ascompared to the RSV NAb titers. As indicated in the methods, guinea pigcomplement was included in the neutralization assay for RSV but wasexcluded from the HPIV1 neutralization assay as it neutralizes HPIV1.The lack of complement may be the reason for generally reduced HPIV1 NAbtiters. The rHPIV1 C^(Δ170)-F2 and -F3 viruses did not induce detectablelevels of HPIV1 NAbs. Only the rHPIV1 C^(Δ170) empty vector and therHPIV1 C^(Δ170)-F1 induced detectable HPIV1-specific NAb responses,which were at titers that were not statistically different from eachother. This was contrary to the expectation because the expression ofall vector proteins was significantly reduced for rHPIV1 C^(Δ170)-F1(FIGS. 38A and 38B). It also had a lower shut off temperature by 1 C ascompared to the F2 or F3 viruses and it replicated in vivo at levelssimilar to those of F3 virus. Thus, the rHPIV1 C^(Δ170)-F1 was expectedto induce similar or weaker HPIV1 specific NAb response as compared tothe -F3 virus. However, the poor HPIV1 immunogenicity of the C^(Δ170)-F2was consistent with its reduced expression of F and HN proteins (FIG.38B) and poor replication in the URT and LRT (FIG. 40). The lack ofHPIV1 NAb response was unexpected for the C^(Δ170)-F3 virus. Itexpressed all HPIV1 proteins at levels similar to those of the emptyvector (FIGS. 38A and 38B), replicated similarly, and had the shut offtemperature similar to that of the empty vector but showed no detectableNAb response.

All of the rHPIV1 L^(Y942A) viruses, including the empty vector, did notinduce detectable levels of HPIV1 specific NAbs. All rHPIV1 L^(Y942A)viruses expressed reduced levels of HPIV1 proteins in vitro (FIGS. 38Aand 38C) and showed poor or no replication in vivo. Consistent withthis, these viruses also showed lack of immunogenicity against RSV.Their poor immunogenicity is likely a result of their strong tsphenotype (FIG. 43) and lack of in vivo replication (FIG. 40).

Immunization with rHPIV1 Vectors Expressing RSV F Provides ProtectionAgainst Wt RSV Challenge.

Hamsters were immunized with rHPIV1 expressing RSV F as described aboveand were challenged on day 30 post-immunization with 10⁶ PFU of wt RSV.Hamsters were euthanized on day 3 post-challenge and RSV titers in thenasal turbinates and lungs were determined by plaque assay on Vero cellsto assess the protection against challenge RSV replication (FIGS. 41Aand 41B). The protection provided by vaccine candidates against RSVinfection and replication correlated with their ability to induce RSVspecific serum NAb. The rHPIV1 C^(Δ170)-F1 and -F3 provided significantprotection against RSV replication and showed significantly reduced(p<0.0001 and p<0.05, respectively) RSV titers in the URT as comparedwith the rHPIV1 C¹⁷⁶ empty vector, with the -F2 virus having no effect.In the lungs, although all three rHPIV1 C^(Δ170)-F1, -F2, and -F3viruses reduced the mean RSV titers as compared to the rHPIV1 C^(Δ170)empty vector, only the RSV reduction by -F1 virus was statisticallysignificant (p<0.05). As expected, the rB/HPIV3-F2 provided significantprotection against RSV challenge in the URT (P<0.0001) and lungs(p<0.05), which was consistent with a previous report (Liang, et al.2014. J virology 88:4237-4250, and Example 1).

Statistical comparison of the rHPIV1 vaccine candidates with therB/HPIV3-F2 virus showed that the protection provided by rHPIV1C^(Δ170)-F1 was statistically similar to that of rB/HPIV3-F2 both in theURT and lungs. The rHPIV1 C^(Δ170)-F1 replicated in hamsters to titerssimilar to those of rB/HPIV3-F2 on day 3 post-infection but demonstratedsignificantly reduced replication on day 5 post-infection suggestingthat the rHPIV1 C^(Δ170)-F1 may be sufficiently attenuated in vivo. Thisalong with its ability to protect against RSV challenge similar to thatof rB/HPIV3-F2 also indicated that rHPIV1 C^(Δ170)-F1 has desirablefeatures of attenuation and immunogenicity and should be furtherdeveloped as a live attenuated RSV vaccine candidate.

The rHPIV1 L^(Y942A)-F1, -F2, and -F3 viruses did not provide protectionagainst RSV challenge in the URT and lungs and showed challenge RSVloads similar to that of the rHPIV1 L^(Y942A) empty vector. This wasconsistent with their lack of in vivo replication and immunogenicityagainst RSV.

Discussion

Various attenuated versions of rHPIV1 bearing attenuating and/or ismutations have been previously developed that were immunogenic inrodents and/or non-human primates. Two attenuated rHPIV1 backbonescontaining either the C^(Δ170) or the L^(Y942A) mutation involving adeletion of 6 and substitution of 3 nucleotides, respectively, wereassayed. RSV F was inserted at the first, second, or third genomeposition of each backbone with the aim to identify a construct that isappropriately attenuated and yet sufficiently immunogenic and protectsagainst wt RSV challenge. All rHPIV1 viruses expressing RSV F weresuccessfully rescued by reverse genetics. Growth kinetics in Vero andLLC-MK2 cells indicated that all viruses grew to very similar andstatistically indistinguishable final titers determined at 7 dayspost-infection (FIG. 37). However significant differences in replicationwere observed on day 2 in both cell lines. The L^(Y942A) mutation seemedto have a stronger attenuating effect than the C^(Δ170) mutation atleast in Vero cells. Contrary to the expectation, the rHPIV1C^(Δ170)viruses were not attenuated in type I interferon (IFN-I) competentLLC-MK2 as compared to IFN-I deficient Vero cells. Among the rHPIV1C^(Δ170) viruses expressing RSV F, significant attenuation as comparedto the wt HPIV1 was observed for the -F1 and -F2 viruses in Vero (FIG.37A) and for -F1 in LLC-MK2 cells (FIG. 37B), whereas the -F3replication was similar to the empty vector and wt HPIV1. These datasuggest that insertion of RSV F closer to the 3′ proximal positions in-F1 or -F2 viruses may be attenuating but this effect was transient andall viruses grew to similar final titers (FIGS. 37A and 37B). All rHPIV1L^(Y942A) backbone vectors including -F1, -F2 and -F3 grew to similarfinal titers on day 7. However, they also demonstrated reducedreplication in both Vero and LLC-MK2 cells on days 2, 3, and 4 but weresimilar to wt HPIV1 between 5-7 days. The RSV F insert inducedattenuation was much greater in magnitude for the rHPIV1L^(Y942A) ascompared to the rHPIV1 C^(Δ170) vectors (FIG. 37D) indicating that RSV Finsertion has a stronger attenuating effect on a backbone that isalready significantly attenuated.

All viruses were examined for their ability to express RSV F protein byWestern blot analysis of infected Vero cells incubated at a permissivetemperature of 32° C. The expression of the HPIV1 N, P, F, and HNproteins was also evaluated to determine the effect of RSV F insertionat various positions on the vector protein expression. Unexpectedly, apolar gradient of RSV F expression was not observed for the rHPIV1C^(Δ170) vectors. The rHPIV1 C^(Δ170)-F1 demonstrated significantlydecreased expression of all vector proteins tested. Similarly, with theexception of N protein, the rHPIV1 C^(Δ170)-F2 also showed reducedexpression of P, F, and HN proteins (FIG. 38A and 38B). Consistent withthis both the -F1 and -F2 viruses showed reduced replication in Verocells early during infection (FIG. 37A). These data suggest that for F1and F2 viruses, the combined effect of reduced vector protein synthesisand the consequent attenuated replication might be responsible forreduced RSV F expression and a lack of its polar gradient. rHPIV1C^(Δ170)-F3 showed a modest reduction in RSV F expression as compared toF1 and F2, likely due to its distal genome location. The RSV Fexpression profile was very different for the rHPIV1 L^(Y942A) vectorsthat demonstrated a strong polar gradient of expression (FIGS. 38A and38C) with the F1 virus showing significantly higher RSV F expression ascompared to F2 and F3. As anticipated, insertion of RSV F in the firstposition significantly reduced the N protein expression for the F1vector while the P, F and HN proteins were unaffected and had expressionsimilar to that of their empty vector counterpart (FIGS. 38A and 38C).The rHPIV1 L^(Y942A)-F2 showed very poor expression of RSV F as well asall vector proteins (FIGS. 38A and 38C). As stated above, this virus wasthe most difficult to rescue due to its highly attenuated phenotype andthe accumulation of mutations during rescue. However, the virus finallyused in the experiments had no adventitious mutations confirmed bygenome sequencing. Therefore, it appears that insertion of RSV F at theF2 location in rHPIV1 L^(Y942A) backbone seems to be quite detrimentalwith the virus showing drastically significant reduction of all vectorproteins tested including RSV F. One non-limiting explanation for thisresult is that this is a combined effect of insert position and thehighly attenuated backbone because such effects were not observed forrHPIV1 C^(Δ170)-F2. Referring to its growth kinetics (FIGS. 37C-37D),the rHPIV1L^(Y942A)-F2 was the most attenuated of all the rHPIV1L^(Y942A) vectors. Thus, it seems that an overall reduction of vectorprotein synthesis significantly reduced its replication resulting inreduced RSV F expression. The rHPIV1 L^(Y942A)-F3 virus expressed vectorproteins at levels similar to the empty vector but showed a >10-foldreduction in RSV F expression as compared to F1 (FIGS. 38A and 38C).This indicates that RSV F insertion at the third genome position did notnegatively affect vector protein expression (FIGS. 38A and 38C) butdemonstrated poor RSV F expression likely due to its distal location inthe polar transcriptional gradient.

Native RSV F causes plasma membrane fusion of the neighboring infectedcells resulting in syncytia formation. The HPIV1 vector F protein doesnot cause syncytium formation, which therefore could be used as anindicator of RSV F functionality and native form as well as quantity ofexpression. Formation of syncytia by RSV F expressed from rHPIV1C^(Δ170) or L^(Y942A) vectors was evaluated in Vero and LLC-MK2 cells.Extensive syncytia formation showing fusion of the majority of cells inthe monolayer was observed with the rHPIV1 C^(Δ170-)F1 and -F2 as wellas with the rHPIV1 L^(Y942A)-F1 (FIG. 39) suggesting that therecombinant RSV F protein is functional and presumably in a nativeconformation. The rHPIV1 C^(Δ170-)F3 and L^(Y942A)-F2 and -F3 did notshow apparently obvious syncytia formation. The extent of syncytiaformation was consistent with the level of RSV F expression detected byWestern blot (FIGS. 38A-38C).

Since the ts phenotype plays an important role in virus replication invivo and may determine immunogenicity, the ts phenotype of the vectorsexpressing RSV F was evaluated. In contrast to the wt HPIV1 that is notts even at 40° C., the rHPIV1 C^(Δ170) and L^(Y942A) backbones were tsat 40° C. and 36° C., respectively (FIG. 43). Important to note is thatthe insertion of RSV F in the rHPIV1 C^(Δ170) at F1 and the rHPIV1L^(Y942A) at F1 or F2 positions lowered the shut off temperature by 1°C. thus making them slightly more ts. This effect may not be unique toHPIV1 because similar effect was observed on RSV F insertion into therB/HPIV3 vector in a previous study Liang, et al. 2014. J virology88:4237-4250). The enhancement of ts phenotype was observed for rHPIV1L^(Y942A)-F2 but not for rHPIV1 C^(Δ170)-F2 indicating that theinsertion of foreign gene may enhance the ts phenotype of a virus thatis already significantly ts.

The rHPIV1 vectors were evaluated in hamsters to assess theirreplication and immunogenicity. All of the rHPIV1 L^(Y942A) viruses wereover-attenuated and virus replication was undetectable in the URT andlungs of the majority of animals. This is consistent with their highlyts phenotype with a shut off temperature of 35-36 C. Failure toreplicate seems not to be due to RSV F insertion but is likely an effectof their ts phenotype because even the empty vector did not replicate inthe lungs and had poor replication in the URT. The rHPIV1 C^(Δ170)-F1,-F2, and -F3 were overall highly restricted and undetectable in thelungs while the empty rHPIV1 C^(Δ170) vector did show low levelreplication, which was significantly lower than that of the wt HPIV1,suggesting that the presence of RSV F insert had an additionalattenuating effect on the already attenuated rHPIV1 C^(Δ170) backbone inthe lungs. In contrast to the lungs, all rHPIV1 C^(Δ170) vectorsreplicated well in the URT of all animals. The F1 and F2 viruses, butnot F3, were significantly attenuated as compared to wt HPIV1, with F2being more attenuated than F1. This was unexpected because in generalinsertion closer to the 3′ end of the genome results in higherattenuation. This was also consistent with the relatively slower earlygrowth of F1 and F2, but not F3, in vitro (FIG. 37) indicating thatinsertion of RSV F, in the F1 and particularly at F2 position, has anadditive attenuating effect. The F1 virus appears to have the desireddegree of attenuation, whereas the F2 and F3 viruses are over- andunder-attenuated, respectively.

Attaining an optimal balance between attenuation and immunogenicity is achallenge with live attenuated vaccines. To assess if the attenuatedrHPIV1 vectors were sufficiently attenuated, their replication wascompared with that of the rB/HPIV3-F2, a leading RSV vaccine vectorexpressing RSV F from the second genome position (Liang, et al. 2014. Jvirology 88:4237-4250). The replication of rHPIV1 C^(Δ170)-F1, -F2, or-F3 on day 3 and 5 was either statistically similar to or significantlylower than that of the rB/HPIV3-F2 in both the URT and lungs. Thissuggests that the C^(Δ170) mutation together with the insertion of RSV Fappear to have achieved the desired level of attenuation, at least inthis animal model, such that all three vectors are highly restricted inthe lungs but do demonstrate attenuated replication similar torB/HPIV3-F2 in the URT that will be needed for immunogenicity.

A difficulty encountered by RNA virus vectored vaccines is theinstability of the foreign antigen gene in vivo (Yang, et al. 2013.Vaccine 31:2822-2827). Mutations are generated due to the error pronepolymerase, and the lack of a need to maintain the expression of theinsert. Any mutations in the foreign antigen acquired due to infidelityof the RNA dependent RNA polymerase could be positively selected as theyprovide a selective advantage. To determine the stability of RSV Fexpression in vivo after immunization, viruses recovered fromrespiratory tissues of hamsters were analyzed by fluorescent doublestaining plaque assay. This could only be performed for HPIV1C^(Δ170)-F1, -F2, and F3 viruses that showed detectable replication inthe URT (FIG. 44). For majority of the samples, stable RSV F expressionwas observed for all three viruses, except one HPIV1 C^(Δ170)-F1 samplefor which RSV F expression was detected for 98% plaques. These dataindicated that the rHPIV1 C^(Δ170) vectors maintain a stable expressionof RSV F during in vivo replication.

Immunogenicity of the rHPIV1 vectors was evaluated by performing thePRNT₆₀ assay. The assay for RSV was performed in the presence of guineapig complement, which was excluded from the HPIV1 neutralization assaybecause of the direct neutralization of HPIV1 by the complement alone.The rHPIV1 C^(Δ170)-F1, -F2, and -F3 induced RSV neutralizing antibodiesat a PRNT₆₀ (log₂) titer of 7.3, 4.7, and 6.7, respectively (FIG. 45).Although F1 demonstrated the highest antibody titer, it wasstatistically similar to that induced by F2 and F3. These titers weresignificantly lower than those induced by rB/HPIV3-F2 or wt RSV controlswhich could be a result of their overall relatively reduced replicationboth in the URT and lungs. The rHPIV1 L^(Y942A)-F1, F2, and F3 virusesdid not induce a detectable RSV or HPIV1 neutralizing antibody response,which was consistent with their lack of replication in vivo. Only therHPIV1 C^(Δ170)-F1 and not the F2 or F3, induced detectable HPIV1neutralizing antibodies. This was unexpected because the F2 and F3 didshow replication in hamsters (FIG. 40) and induced RSV NAbs. Asindicated above guinea pig complement, known to enhance the PRNT₆₀ readout, could not be included in the HPIV1 neutralization assay. Theoverall low HPIV1 antibody titers and lack of a detectable response,even for viruses that replicated in hamsters, could be due to weakersensitivity of the assay lacking complement.

To determine the protective efficacy of the vectors against RSVinfection, all immunized hamsters were intranasally challenged at 30days post-immunization with a high dose (10⁶ pfu) of wt RSV per animal.Protection against challenge was assessed by determining RSV replicationin the nasal turbinates and lungs (FIGS. 41A and 41B). Protectiondirectly correlated with the immunogenicity (FIG. 45). Highly consistentwith the RSV NAb titers, rHPIV1 C^(Δ170)-F1 was more protective than F3and no protection was afforded by F2. The protection was statisticallysignificant for F1 in both the URT and lungs and for F3 in the URT only.Importantly, rHPIV1 C^(Δ170)-F1 afforded protection in the URT and lungsthat was statistically indistinguishable from rB/HPIV3-F2. This wassomewhat unexpected because the rHPIV1 C^(Δ170)-F1 induced significantlylower RSV NAb titer than that of the rB/HPIV3-F2 suggesting that therelatively lower NAb titer was sufficient to achieve similar protection.This interpretation is supported by the evidence that RSV replication inthe respiratory tract of cotton rats could be reduced if the serum NAbtiter was 1:100 or greater (Prince, et al. 1985. J Virol 55:517-520).Again, consistent with their lack of sero-conversion, rHPIV1L^(Y942A)-F1, F2, and F3 did not provide any protection againstchallenge and had RSV loads similar to that of the empty vector. Thesedata clearly show that the rHPIV1 L^(Y942A) vectors were over-attenuateddue to their is phenotype and did not replicate in the hamstersresulting in a lack of immunogenicity both for RSV and HPIV1. Incontrast, the rHPIV1 C^(Δ170) vectors, although highly restricted in thelungs, did replicate in the URT. Among the vectors tested in this study,the rHPIV1 C^(Δ170)-F1 appears to be adequately attenuated and yetsufficiently immunogenic against RSV. It is recognized that constructsthat appear to be over-attenuated in hamsters may perform more suitablywhen evaluated in a more permissive primate host.

In summary, this example identifies the rHPIV1 C^(Δ170) as a promisingattenuated backbone suitable for expressing RSV F antigen from aninserted gene. It was also systematically determined that the F1 (pre-N)genome position of the rHPIV1 C^(Δ170) vector was preferred among thepositions tested for inserting RSV F. This study also demonstrated thatthe rHPIV1 C^(Δ170)-F1 is a promising vaccine candidate and possessesseveral desirable features: (i) it is based on a backbone wellcharacterized for attenuation in non-human primates (3), (ii) itreplicated in Vero cells to final titers similar to that of wt HPIV1, anessential feature for vaccine manufacture, (iii) insertion of RSV Fattenuated it slightly more than the empty C^(Δ170) backbone to a levelsimilar to rB/HPIV3-F2, (iv) the construct was stable after in vivoreplication and maintained RSV F expression, and (v) it was the mostimmunogenic HPIV1 vector inducing the highest RSV and HPIV1 neutralizingantibody titer and was also the most protective against a wt RSVchallenge.

The findings indicate that it is possible to use an HPIV1 vectorexpressing RSV F as a bivalent vaccine for mucosal immunization againstRSV and HPIV1. An HPIV1 vectored RSV vaccine could be used either as aprimary RSV vaccine or to boost immunity primarily induced by a liveattenuated RSV. The HPIV1 vectored RSV vaccine approach would obviatethe inherent problems associated with developing attenuated RSV strainsand may facilitate RSV immunization programs even in resource-limitedsettings.

Example 3 Development of rHPIV1-C^(Δ170) Vectors Expressing OptimizedVersions of RSV F Protein

This example presents assays showing that a gene encoding a modified RSVF ectodomain can be inserted into a HPIV1 vector backbone to produce arecombinant virus that expresses RSV F ectodomain on its envelope, isattenuated, infective, and can induce a protective antibody response.The F2 position also was identified as an effective insertion site.These findings indicate that:

1. The operational boundaries of the HPIV1 F TMCT domains have beenidentified (FIG. 60).

2. The HPIV1 F TMCT domain can be added to a recombinant RSV Fectodomain, e.g., HEK/GS-opt/DS-Cav1 to achieve a protein that isefficiently expressed at the cell surface and reactive with anti-RSV-Fantibodies.

3. RSV F containing the HPIV1 F TMCT (HEK/GS-opt/DS-Cav1/H1TMCT) wasefficiently packaged into the HPIV1 vector particle (i.e., at a higherlevel per μg virion protein than RSV, FIG. 64). This was completelycontrary to expectations based on previous studies with Sendai virus(the murine relative of HPIV1, and hence presumably a close predictivemodel), in which RSV F containing the Sendai virus CT or TMCT waspackaged efficiently into the particle only if the endogenous Sendaivirus F protein was deleted (Zimmer et al 2005 J Virol 79:10467-10477).

3. HEK/GS-opt/DS-Cav1 and HEK/GS-opt/DS-Cav1/H1TMCT forms of the RSV Fprotein can be inserted into the first or second gene positions to yieldvector constructs that stably express RSV F, replicate efficiently invitro (FIG. 63), and are efficiently incorporated into the vectorparticle (when HPIV1 F TMCT is present, FIG. 64).

4. Unexpectedly, while either the F1 or F2 positions are efficient inexpressing RSV F protein that is fusogenic (e.g., HEK/GA-opt, Example2), the F2 position was particularly efficient for intracellularexpression of RSV F that was non-fusogenic (e.g., HEK/GS-opt/DS-Cav1).

5. The rHPIV1-C^(Δ170)-F2/HEK/GS-opt/DS-Cav1 andrHPIV1-C^(Δ170)-F2/HEK/GS-opt/DS-Cav1/H1TMCT constructs were identifiedas ones that efficiently expressed RSV F protein and, particularly inthe latter case, efficiently incorporated RSV F into the vectorparticle.

HEK/GS-opt/DS-Cav1.

In the present Example, the rHPIV1-C^(Δ170) vector was used to expressfurther-modified versions of the RSV F protein. All inserts contain RSVF that was codon optimized by Genescript (GS-opt) for human expressionand had two HEK amino acid assignments, i.e., Glu and Pro at residues 66and 101. In addition, the HEK/GS-opt RSV F protein contained thestabilized pre-fusion mutations DS (S155C and S290C) and Cav1 (S190F,and V207L) to stabilize the RSV F prefusion head and antigenic site Øthat have been shown to be responsible for the preponderance ofRSV-neutralizing antibodies (McLellan et al 2013 Science 342:592-598).

HPIV1 TMCT.

In addition, a version of the HEK/GS-opt/DS-Cav1 RSV F protein was madein which its TMCT domain was replaced by that of HPIV1 F protein. Thecomposition of the HPIV1 TM and CT domains had not been previouslydetermined. FIG. 60 indicates the TM and CT domains of RSV F protein(top line) and HPIV1 F protein (second line), and shows a chimera inwhich the predicted ectodomain of RSV F protein was attached to thepredicted TMCT domains of HPIV1 F protein. This was done with the goalof increasing the incorporation of the RSV F protein into the HPIV1vector, on the premise that the HPIV1 F-specific TMCT domain wouldinteract more efficiently with the other vector proteins during viralassembly, and would facilitate incorporation of the chimeric RSV Fprotein.

rHPIV1-C^(Δ170) Vector Constructs.

FIGS. 61 and 62 show constructs in which the rHPIV1-C^(Δ170) vector fromExample 2 was used to accept either of two inserts, placed in the firstgene position (F1): expressed RSV F HEK/GS-opt/DS-Cav1, yieldingrHPIV1-C^(Δ170)-F1/HEK/GS-opt/DS-Cav1 (FIGS. 61 and 62, top construct);or RSV F HEK/GS-opt/DS-Cav1/H1TMCT, yieldingrHPIV1-C^(Δ170)-F1/HEK/GS-opt/DS-Cav1/H1TMCT (bottom construct). Notethat these two constructs differ only in that the RSV F in the secondconstruct has TMCT from HPIV1 F protein. FIG. 62 shows two parallelconstructs in which either insert was placed in the second gene position(F2) of the rHPIV1-C^(Δ170) vector. All viruses were designed to keepthe hexameric genome nucleotide length (rule of six) (Kolakofsky et al1998 J Virol 72:891-899). Each vector gene maintained its wild typehexamer phasing: the F1 and F2 inserts had the hexamer phasing of the Nand P aeries, respectively.

Recovery of Viruses.

The four constructs were rescued by co-transfecting BHK BSR T7/5 cells(baby hamster kidney cells that constitutively express T7 RNA polymerase(Buchholz et al 1999 J Virol 73:251-259)) with each of the full-lengthanti-genome plasmids and three expression plasmids expressing the HPIV1N, P, and L proteins. Double staining plaque assays detectingco-expression of HPIV1 and RSV F proteins were performed to determinethe stability of RSV F expression. The vast majority of plaquesefficiently expressed RSV F protein. These same four preparations weresubjected to consensus sequence analysis by automated sequencing (whichanalyzed each genome in its entirety except for 22 and 26 nucleotides atthe 3′ and 5′ end, respectively, which were obscured by primers). Theviruses were found to be free of adventitious mutations detectable bythis method.

Multi-Cycle Replication In Vitro.

The four recovered viruses (i.e., HEK/GS-opt/DS-Cav1, with or withoutH1TMCT, in the F1 or F2 position) were evaluated for multi-cyclereplication in vitro by infecting Vero cells at an MOI of 0.01 TCID₅₀per cell with each virus in triplicate and collecting culturesupernatant every 24 h for 7 days. Virus titers in the collected sampleswere determined by serial dilution on LLC-MK2 cells and hemadsorptionassay. All vectors with RSV F insert were relatively attenuated ascompared to wt HPIV1 and rHPIV1-C^(Δ170) and grew to final titers around7.0 TCID₅₀/mL (FIG. 63). The F1/DS-Cav1 and F1/DS-Cav1/H1TMCT (note, the“HEK/GS-opt” part of the name may be omitted in the text and figures forthe sake of brevity) replicated slower than the F2/DS-Cav1 andF2/DS-Cav1/H1TMCT on day 1 and 2 post-infection (p.i.) (during theexponential phase of replication) but reached high titers by day 7(harvest day) that were similar to F2/DS-Cav1 and F2/DS-Cav1/H1TMCT.Thus, all constructs grew to high final titers that are amenable tovaccine manufacture in Vero cells.

Incorporation of Proteins into Vector Virions.

The set of four constructs along with wt HPIV1, and rHPIV1-C^(Δ170)empty vector were grown in LLC-MK2 cells and purified by sucrosegradient centrifugation. wt RSV was propagated in Vero cells followed bysucrose gradient purification and was included as a control.Approximately 1 μg of each sucrose-purified virus was lysed in RIPAbuffer, reduced, denatured and subjected to SDS-PAGE and Western blotanalysis. RSV F protein and HPIV1 proteins (N, F, and HN) were detectedwith mouse monoclonal (Abcam) and rabbit polyclonal peptide-specificantibodies (see Example 2), respectively, along with their correspondinginfrared dye-conjugated secondary antibodies as previously described(Example 2; Mackow et al 2015 J Virol 89:10319-10332) (FIG. 64).Analysis of the F1/DS-Cav1 and F2/DS-Cav1 purified virions, whichexpressed full length RSV F from the 1^(st) and 2^(nd) position,respectively, demonstrated no detectable packaging of RSV F in the HPIV1vector virions (FIG. 64, top panel, lanes 3 and 5. In contrast, both theF1/DS Cav1-H1TMCT and F2/DS Cav1-H1TMCT virions (FIG. 64, lanes 4 and 6)showed considerable incorporation, which was higher for the F1/DSCav1-H1TMCT than F2/DS Cav1-H1TMCT. Interestingly, both H1TMCT versionsshow higher amounts of RSV F packaged in the virions as compared to wtRSV (FIG. 64, lane 7), per μg of virion protein.

In the same experiment, the incorporation of the HPIV1 N, HN, and Fproteins into the HPIV1 vector virions was also evaluated (FIG. 64). Thevirion incorporation of HPIV1 N protein was little affected byexpression of any of the inserts (FIG. 64, second panel from the top).HN protein was reduced for F1/DS-Cav1/H1TMCT, F2/DS-Cav1, andF2/DS-Cav1/H1TMCT constructs (FIG. 64, third panel from the top, lanes4, 5, and 6). Virion incorporation of HPIV1 F protein was reduced forF2/DS-Cav1 and F2/DS-Cav1/H1TMCT (FIG. 64, fourth panel from the top,lanes 5 and 6). Note that the antiserum for the HPIV1 F protein had beenraised against a peptide containing the last 18 amino acids of the CTdomain of the HPIV1 F protein, and thus this antiserum detected, inaddition to the HPIV1 F₀ and F₁ proteins, the forms of RSV F proteincontaining the HPIV1 F protein TMCT, namely F1/DS-Cav1/H1TMCT andF2/DS-Cav1/TMCT. The identity of the RSV F band was confirmed byco-staining with the anti-RSV F monoclonal antibody; this confirms thatRSV F is indeed expressed with HPIV1 F TMCT domain.

Intracellular Protein Expression.

Next, the intracellular expression of RSV F protein and HPIV1 vectorproteins in vector-infected cells was evaluated. Vero cells wereinfected at an MOI of 10 TCID₅₀ per cell with each construct andincubated at 32° C. for 48 h. Cellular proteins were harvested by directlysis of the monolayer with 1×LDS buffer, reduced, denatured, andsubjected to SDS-PAGE and Western blot analysis (FIG. 65). RSV F andHPIV1 proteins were detected by using the same antibodies as describedin FIG. 64. All constructs were able to express RSV F protein ininfected cells; F2/DS-Cav1/H1TMCT had the highest expression followed byF2/DS-Cav1, F1/DS-Cav1/H1TMCT, and F1/DS-Cav1 (FIG. 65, lanes 4, 3, 2,and 1). Contrary to the expectation that insertion of the RSV F insertinto the first gene position would result in the highest levels ofexpression (due to the transcriptional gradient), the F1/DS-Cav1 andF1/DS-Cav1/H1TMCT viruses (FIG. 64, lanes 1 and 2) had reduced RSV Fexpression as compared to the F2 viruses (lanes 3 and 4). As alreadynoted, the F1 viruses replicated somewhat more slowly during exponentialgrowth than the F2 viruses (FIG. 63), which likely contributed to thereduced RSV F expression. The low level of intracellular expression ofRSV F in cells infected with the F1 viruses is in contrast to the virionincorporation profile (FIG. 64) that shows higher incorporation of RSV Ffor F1/DS-Cav1/H1TMCT as compared to F2/DS-Cav1/H1TMCT (lane 4 versus6). In both F1 and F2 positions, DS-Cav1 with TMCT was expressed atsomewhat higher level than DS-Cav1 alone, suggesting TMCT may enhancethe RSV F expression by making the protein or mRNA transcript morestable. Similar effect was also observed with RSV F with BPIV3 F TMCT inVero cells infected by B/HPIV3 vectors (FIG. 50A). It also is noteworthythat the intracellular expression of the HPIV1 N, P, F, and HN proteinsalso was very low for the F1 viruses compared to the F2 viruses (FIG.65, second, third, and fourth panels, lanes 1 and 2 versus 3 and 4).This reveals very low gene expression for the F1 viruses (FIG. 65, lanes1 and 2), although the RSV F protein that is made apparently was veryefficiently incorporated into the HPIV1 virion in the case of RSV Fbearing the HPIV1 TMCT (FIG. 64, lane 4).

Reduced RSV F expression by the F1 viruses in this present experiment isin contrast to the results reported in Example 2 involving expression ofan HEK/GA-opt form of the RSV F protein from the first gene position ofthis same vector. In that case, intracellular expression of the RSV Fprotein was efficient and was very similar for the F1 versus the F2construct, whereas the expression of the HPIV1 vector proteins wassomewhat reduced for F1 versus F2, but not drastically so (FIG. 38A,first two lanes at the left, and FIG. 38B). The difference may be thatthe version of RSV F expressed in FIG. 38 was fusogenic, and this mayhave helped the virus spread more efficiently through the cell monolayerand promote a more efficient infection resulting in greater proteinsynthesis, whereas the F protein expressed in FIG. 64 was the DS-Cav1version that was frozen in the prefusion conformation and thus would benon-fusogenic. While the constructs expressing DS-Cav1 may not havespread efficiently, each infected cell might have made more RSV Fprotein, and this could account for the higher level of virionincorporation. However, since a relatively high MOI of infection wasused in each experiment (MOI of 5), and thus the great majority of cellsshould have been infected and spread may not have been a major factor.Therefore, the reduced intracellular expression of the DS-Cav1 form ofthe F protein from the F1 position was unexpected and points to the F2position as likely being the more suitable for vector use.

Example 4 Development of wt rHPIV3 Strain JS Vectors ExpressingOptimized Versions of RSV F Protein

As discussed above, the addition of the TMCT of the BPIV3 F protein(B3TMCT) to the RSV F HEK/GA-opt/DS construct resulted in substantialattenuation of the rB/HPIV3 vector in rhesus monkeys (FIG. 29). Therealso was evidence of attenuation in hamsters due to the B3TMCT (FIG.51). This raised the possibility that this would render the rB/HPIV3vector over-attenuated. One solution would be to use a less attenuatedvirus, such as wt HPIV3, as vector. In addition, if an HPIV3 vector wasused (i.e, all of the backbone genes were derived from HPIV3), thiswould provide a complete complement of HPIV3 proteins as antigens forcellular immunity against HPIV3, and thus an HPIV3-based vector might bemore protective against HPIV3 than an rB/HPIV3-based vector. Thefindings discussed in this example indicate that, unexpectedly, thestability of expression of the RSV F insert F1-HEK/GS-opt/DS-Cav1/H3TMCTappeared to be more stable than its version lacking TMCT, namelyF1-HEK/GS-opt/DS-Cav1. This is contrary to expectations, sinceincorporation into the vector particle might have been expected to placean added selective pressure against maintenance of RSV F expression, butthis was not observed.

Wt rHPIV3 JS Strain Vector.

The wild type (wt) recombinant (r)HPIV3 JS strain (which also was thesource of the F and HN genes in rB/HPIV3) was selected as vector. Thebiological version of this virus was previously shown to be naturallyattenuated in adults (Clements et al. J Clin Microbiol 29:1175-1182,1991) compared to a previously-evaluated strain (Kapikian et al 1961JAMA 178:123-127), presumably due to one or more adventitiousattenuating mutations that remain to be identified. The recombinantversion of this virus (rHPIV3) has two mutations in HN (A263T and T370P)that were introduced when the HPIV3 reverse-genetic system wasestablished. In the present study, the rHPIV3 vector was modified tocontain the 263T and 370P amino acid assignments in the HN protein thathad been found to prevent large plaque formation by the rB/HPIV3 vector(the mutations were shown in detail in FIG. 58B). In addition, therHPIV3 vector was modified by the creation of a unique BlpI site atpositions 103-109 (FIG. 67A, top construct), for insertion of RSV F (orpotentially any other insert) in the first gene position, or thecreation of a unique AscI site at positions 1675-1682 (FIG. 67B, topconstruct), for insertion of RSV F in the second gene position.

HEK/GS-opt/DS-Cav1+/− H3TMCT.

The wt rHPIV3 JS vector was used to express the RSV F HEK/GS-opt/DS-Cav1protein as is or with the further modification in which the TMCT domainsof the RSV F protein was replaced by that of the HPIV3 F protein (calledH3TMCT, FIG. 66). The activity of the TM and CT domains of HPIV3 Fprotein (H3TMCT) in the context of the RSV F protein was not known. FIG.66 also shows a chimera in which the H3TMCT was introduced into the RSVF protein.

Examples of four rHPIV3-based constructs expressing RSV F are shown inFIG. 67. Specifically, the examples are:

HEK/GS-opt/DS-Cav1 inserted into the first gene position (F1) (FIG.67A);

HEK/GS-opt/DS-Cav1/H3TMCT inserted into the first gene position (F1)(FIG. 67A);

HEK/GS-opt/DS-Cav1 inserted into the second gene position (F2) (FIG.67B);

HEK/GS-opt/DS-Cav1/H3TMCT inserted into the second gene position (F2)(FIG. 67B).

Note that these RSV F ORFs are the same as were inserted into therHPIV1-C^(Δ170) vector in FIGS. 61 and 62, except that the TMCT in FIG.67 is H3TMCT derived from HPIV3 F protein. In addition, each RSV Finsert in FIG. 67 was under the control of HPIV3 transcription signalsfor expression as a separate mRNA. Nucleotide numbering (FIG. 67) isrelative to the complete antigenome RNA sequence of each finalconstruct. All viruses were designed to maintain the hexameric genomelength and wild type hexamer phasing; the F1 and F2 inserts had thehexamer phasing of the original N and P genes (normally in the first andsecond gene positions), respectively. All inserts were syntheticallyderived (Genscript),

Recovery of Viruses and Double Staining Analysis.

The viruses were rescued by co-transfecting BHK BSR T7/5 cells with eachof the full-length anti-genome plasmid and three expression plasmidsexpressing the HPIV3 N, P, and L proteins. All viruses were successfullyrescued, and two P2 (i.e., second passage) viral stocks were preparedfor each of the four constructs.

Comments on the Results Shown in Examples 1-4

The following examples show that several different PIV vector systems(including rB/HPIV3, rHPIV1, and rHPIV3 JS) can be used to efficientlyexpress the RSV F protein as an added gene. Panels of mutants wereconstructed to systematically evaluate a variety of variables with thevectors (e.g., insert position, level of attenuation, etc.) and the RSVF insert (e.g. prefusion stabilization, packaging into the vectorparticle, etc.), The panels of mutants were subjected to detailedanalysis in cell culture, in hamsters, and in non-human primates toidentify effective mutants with regard to expression level, stability invitro and in vivo, attenuation in vivo, immunogenicity, and protectiveefficacy.

Previous studies had shown that highly fusogenic proteins like RSV F canbe unstable in nonsegmented negative strand viral vectors because theirhigh level of syncytium formation interferes with vector replication:for example, the fusion F protein of measles virus was shown to exhibit“extreme instability” when expressed by the prototype virus vesicularstomatitis virus (VSV) (Quinones-Kochs et al 2001 Virology 287;427-435). Since respiratory syncytial virus is notorious for its highlevel of syncytium formation, and since high levels of syncytiumformation indeed were observed when functional RSV F protein wasexpressed from PIV vectors (e.g., Example 1), it was very surprisingthat this did not interfere with vector replication in vitro. Thus,efficiencies of replication of the disclosed constructs allows forefficient vaccine manufacture in Vero cells. Furthermore, modificationsthat reduced (HEK assignments) or essentially eliminated (DS, DS-Cav1)gross syncytium formation were introduced, which thus would obviate anyproblems due to high levels of syncytium formation.

Multiple specific strategies were demonstrated to increase theexpression of the foreign RSV F gene, including inclusion of HEKassignments, codon optimization, and placement in promoter-proximalpositions. However, the results were not necessarily predictable,illustrating the importance of the extensive experimentation provided inthis disclosure. In addition, while expression from the first geneposition would be expected to yield the highest levels of expression,this was not the case with the HPIV1 vector when it expressed RSV Fprotein that was non-fusogenic due to the DS-Cav1 mutations (FIG. 65).Thus, the nature of the vector and the nature of the insert contributedto yield novel characteristics.

Multiple means of attenuation were investigated, including the use ofthe bovine/human chimera rB/HPIV3, the use of the naturally-attenuatedHPIV3 JS strain, and the use of stabilized point and deletion mutationsexemplified with HPIV1 (C^(Δ170) and LY^(942A)). In addition, presenceof the foreign insert provided attenuation in some circumstances, andthe B3TMCT modification was substantially attenuating. Thus, thereagents and biological characterization provide vectors exhibiting arange of useful attenuation phenotypes. Importantly, the vectors ofinterest were shown to have retained efficient replication and stabilityin Vero cells, necessary for vaccine manufacture.

The stability of expression of the RSV F insert was a major problem witha prior construct (Bernstein et al 2012, Pediatr Infect Dis 31:109-114).However, the modified constructs described in the present disclosurewere substantially stable. Factors such as reducing or inhibitingsyncytium formation likely contributed to stability. In some case,unpredictable factors were identified. For example, the HN gene in therB/HPIV3 vector was found to exhibit substantial instability thatresulted in a large plaque phenotype. However, this problem waseliminated by I263T and T370P substitutions in the HN gene (FIG. 58).Another example of an unexpected finding regarding stability was theincreased stability of RSV F expression with the combination of theHEK/GS-opt/DS-Cav1/H3TMCT construct expressed from the F1 position ofrHPIV3 JS (FIG. 67A). It might have been expected that expression fromthe F1 position would be the most unstable, because of the high level ofexpression, but this was not observed. It also might have been expectedthat packaging of the RSV F protein into the vector also would place aselective pressure for loss of expression, but this was not observed.

Two modifications were shown to be of primary importance for increasingimmunogenicity: namely, mutations DS and Cav1 that stabilize theprefusion conformation of the RSV F protein, and the TMCT modificationthat serves as a packaging signal to direct efficient incorporation ofthe foreign glycoprotein into the vector particle. It was not known ifRSV F that was stabilized in the prefusion conformation would beexpressed efficiently and would be compatible with a virus infection,but this was the case. It also was surprising that the TMCT packagingsignals worked with such a high degree of efficiency of packaging, whichequaled or exceeded that of RSV particles examined in parallel. It alsowas surprising that this highly efficient packaging did not disrupt theproduction of vector particles, such as by displacing the endogenous Fand HN surface glycoproteins.

Particularly unexpected was the finding that the DS and DS-Cav1modifications on the one hand, and the TMCT modification on the otherhand, each provided a very substantial increase in the production ofhigh quality RSV-neutralizing antibodies, which are thought to be themost relevant for protection in vivo. These two types of modificationslikely achieved this effect through different mechanisms: specifically,the DS and Cav1 modifications presumably have their effect bystabilizing the antigenic site Ø, whereas packaging mediated by TMCTlikely provides a tightly-packed highly-repetitive array of RSV Fantigen produced in a particle form, providing for increased antigenpresentation. Importantly, these effects were very evident in non-humanprimates (FIG. 31), yielding reciprocal titers of >250 from a singleprimary dose for complement-independent antibodies. In addition, the twoeffects (DS-Cav1 and TMCT) were additive to some extent.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. A recombinant paramyxovirus, comprising: a viral genomecomprising genes encoding parainfluenza virus N, P, M, F, HN, and Lproteins, and further comprising a heterologous gene encoding a type Imembrane protein comprising a recombinant respiratory syncytial virus(RSV) F ectodomain linked to a transmembrane domain (TM) and cytoplasmictail (CT) of a parainfluenza virus F protein, wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by a nucleotide sequence set forth as SEQ ID NO: 11, 22, or23; and wherein the recombinant paramyxovirus is a recombinanthuman/bovine parainfluenza virus 3 (B/HPIV3), a recombinant humanparainfluenza virus 3 (HPIV3), or a recombinant bovine parainfluenzavirus 3 (BPIV3).
 2. The recombinant paramyxovirus of claim 1,comprising: the recombinant HPIV3, wherein the viral genome comprisesgenes encoding HPIV3 N, P, M, F, HN and L proteins, and wherein the RSVF ectodomain linked to the TM and CT of the parainfluenza virus Fprotein is encoded by the nucleotide sequence set forth as SEQ ID NO:11; the recombinant HPIV3, wherein the viral genome comprises genesencoding HPIV3 N, P, M, F, HN and L proteins, and wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by the nucleotide sequence set forth as SEQ ID NO: 22 or 23;the recombinant BPIV3, wherein the viral genome comprises genes encodingBPIV3 N, P, V, M, F, FIN, and L proteins, and wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by the nucleotide sequence set forth as SEQ ID NO: 11; therecombinant BPIV3, wherein the viral genome comprises genes encodingBPIV3 N, P, V, M, F, FIN, and L proteins, and wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by the nucleotide sequence set forth as SEQ ID NO: 22 or 23;the recombinant B/HPIV3, wherein the viral genome comprises genesencoding HPIV3 F and HN proteins and BPIV3 N, P, V, M, and L proteins,and wherein the RSV F ectodomain linked to the TM and CT of theparainfluenza virus F protein is encoded by the nucleotide sequence setforth as SEQ ID NO: 11; or the recombinant B/HPIV3, wherein the viralgenome comprises genes encoding HPIV3 F and HN proteins and BPIV3 N, P,V, M, and L proteins, and wherein the RSV F ectodomain linked to the TMand CT of the parainfluenza virus F protein is encoded by the nucleotidesequence set forth as SEQ ID NO: 22 or
 23. 3. The recombinantparamyxovirus of claim 1, wherein the heterologous gene encoding therecombinant RSV F ectodomain is the first or second gene downstream of agenomic promoter of the viral genome.
 4. The recombinant paramyxovirusof claim 2, wherein the recombinant paramyxovirus comprises: therecombinant HPIV3, wherein the viral genome comprises, from upstream todownstream, a HPIV3 genomic promoter followed by the genes encoding theHPIV3 N, P, M, F, HN, and L proteins, and wherein the heterologous geneencoding the recombinant RSV F ectodomain is located between the genomicpromoter and the gene encoding the N protein or between the genesencoding the N and the P proteins; the recombinant BPIV3, wherein theviral genome comprises, from upstream to downstream, a BPIV3 genomicpromoter followed by the genes encoding the BPIV3 N, P, M, F, HN, and Lproteins, and wherein the heterologous gene encoding the recombinant RSVF ectodomain is located between the genomic promoter and the geneencoding the N protein or between the genes encoding the N and the Pproteins; or the recombinant B/HPIV3, wherein the viral genomecomprises, from upstream to downstream, a BPIV3 genomic promoterfollowed by the genes encoding the BPIV3 N, P, and M proteins, the HPIV3F and HN proteins, and the BPIV3 L protein, and wherein the heterologousgene encoding the recombinant RSV F ectodomain is located between thegenomic promoter and the gene encoding the N protein or between thegenes encoding the N and the P proteins.
 5. The recombinantparamyxovirus of claim 2, comprising the recombinant B/HPIV3, whereinthe HPIV3 F and HN proteins and BPIV3 N, P, M, and L proteins compriseamino acid sequences at least 90% identical to SEQ ID NOs: 43, 101, 47,48, 49, 52, respectively.
 6. The recombinant paramyxovirus of claim 1,comprising the recombinant B/HPIV3 or the recombinant HPIV3, wherein theviral genome comprises a gene encoding the HPIV3 HN protein, and whereinthe HPIV3 HN protein comprises a threonine and a proline at residues 263and 370, respectively.
 7. The recombinant paramyxovirus of claim 1,comprising: the recombinant HPIV3, wherein the heterologous genecomprises the nucleotide sequence set forth as SEQ ID NO: 11 (GS RSVF_HEK_DS-Cav1_H3TMCT); the recombinant B/HPIV3, wherein the heterologousgene comprises the nucleotide sequence set forth as SEQ ID NO: 11 (GSRSV F_HEK_DS-Cav1_H3TMCT); or the recombinant B/HPIV3, wherein theheterologous gene comprises the nucleotide sequence set forth as SEQ IDNO: 22 (GA RSV F_HEK_DS-Cav1_B3TMCT) or SEQ ID NO: 23 (GS RSV F HEKDS-Cav1_B3TMCT).
 8. The recombinant paramyxovirus of claim 1,comprising: the recombinant HPIV3, and wherein the viral genomecomprises, from upstream to downstream, a HPIV3 genomic promoterfollowed by HPIV3 N, P, M, F, HN, and L genes, and wherein theheterologous gene encoding the recombinant RSV F ectodomain is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain linked to the TM and CT of theparainfluenza virus F protein is encoded by the nucleotide sequence setforth as SEQ ID NO: 11 ; the recombinant HPIV3, and wherein the viralgenome comprises, from upstream to downstream, a HPIV3 genomic promoterfollowed by HPIV3 N, P, M, F, HN, and L genes, and wherein theheterologous gene encoding the recombinant RSV F ectodomain is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by the nucleotide sequence set forth as SEQ ID NO: 11; therecombinant HPIV3, and wherein the viral genome comprises, from upstreamto downstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F,HN, and L genes, and wherein the heterologous gene encoding therecombinant RSV F ectodomain is located between the genomic promoter andthe gene encoding the N protein, and wherein the RSV F ectodomain linkedto the TM and CT of the parainfluenza virus F protein is encoded by thenucleotide sequence set forth as SEQ ID NO: 22 or 23; the recombinantHPIV3, and wherein the viral genome comprises, from upstream todownstream, a HPIV3 genomic promoter followed by HPIV3 N, P, M, F, HN,and L genes, and wherein the heterologous gene encoding the recombinantRSV F ectodomain is located between the genes encoding the N and Pproteins, and wherein the RSV F ectodomain linked to the TM and CT ofthe parainfluenza virus F protein is encoded by the nucleotide sequenceset forth as SEQ ID NO: 22 or 23 ; the recombinant BPIV3, and whereinthe viral genome comprises, from upstream to downstream, a BPIV3 genomicpromoter followed by BPIV3 N, P, M, F, HN, and L genes, and wherein theheterologous gene encoding the recombinant RSV F ectodomain is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain-linked to the TM and CT of theparainfluenza virus F protein is encoded by the nucleotide sequence setforth as SEQ ID NO: 22 or 23; the recombinant BPIV3, and wherein theviral genome comprises, from upstream to downstream, a BPIV3 genomicpromoter followed by BPIV3 N, P, M, F, HN, and L genes, and wherein theheterologous gene encoding the recombinant RSV F ectodomain is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by the nucleotide sequence set forth as SEQ ID NO: 22 or 23 ;the recombinant B/HPIV3, and wherein the viral genome comprises, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and wherein theheterologous gene encoding the recombinant RSV F ectodomain is locatedbetween the genomic promoter and the gene encoding the N protein, andwherein the RSV F ectodomain linked to the TM and CT of theparainfluenza virus F protein is encoded by the nucleotide sequence setforth as SEQ ID NO: 22 or 23 ; the recombinant B/HPIV3, and wherein theviral genome comprises, from upstream to downstream, a BPIV3 genomicpromoter followed by BPIV3 N, P, and M genes, HPIV3 F and HN genes, anda BPIV3 L gene, and wherein the heterologous gene encoding therecombinant RSV F ectodomain is located between the genes encoding the Nand P proteins, and wherein the RSV F ectodomain linked to the TM and CTof the parainfluenza virus F protein is encoded by the nucleotidesequence set forth as SEQ ID NO: 22 or 23 ; the recombinant B/HPIV3, andwherein the viral genome comprises, from upstream to downstream, a BPIV3genomic promoter followed by BPIV3 N, P, and M genes, HPIV3 F and HNgenes, and a BPIV3 L gene, and wherein the heterologous gene encodingthe recombinant RSV F ectodomain is located between the genomic promoterand the gene encoding the N protein, and wherein the RSV F ectodomainlinked to the TM and CT of the parainfluenza virus F protein is encodedby the nucleotide sequence set forth as SEQ ID NO: 22 or 23 ; therecombinant B/HPIV3, and wherein the viral genome comprises, fromupstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P,and M genes, HPIV3 F and HN genes, and a BPIV3 L gene, and wherein theheterologous gene encoding the recombinant RSV F ectodomain is locatedbetween the genes encoding the N and P proteins, and wherein the RSV Fectodomain linked to the TM and CT of the parainfluenza virus F proteinis encoded by the nucleotide sequence set forth as SEQ ID NO: 22 or 23 .9. The recombinant paramyxovirus of claim 1, wherein: the HPIV3 is aHPIV3 JS strain; the HPIV3 comprises I263T and T370P substitutions inthe HN protein; or the B/HPIV3 comprises I263T and T370P substitutionsin the HN protein.
 10. The recombinant paramyxovirus of claim 1, whereinat least 90% of viral particles produced by a host cell infected withthe recombinant paramyxovirus or viral vector comprise a viral envelopecomprising the ectodomain encoded by the heterologous gene.
 11. Therecombinant paramyxovirus of claim 1, wherein the recombinantparamyxovirus is an infectious, attenuated, and self-replicating virus.12. The recombinant paramyxovirus of claim 1, wherein the RSV Fectodomain is present on the viral envelope of the paramyxovirus.
 13. Animmunogenic composition comprising the recombinant paramyxovirus ofclaim 1 and a pharmaceutically acceptable carrier.
 14. A method ofeliciting an immune response to respiratory syncytial virus andparainfluenza virus in a subject comprising administering atherapeutically effective amount of the immunogenic composition of claim13 to the subject.
 15. A nucleic acid molecule comprising the genome ofthe recombinant paramyxovirus of claim
 1. 16. The recombinantparamyxovirus of claim 1, comprising the recombinant B/HPIV3, wherein:the viral genome comprises, from upstream to downstream, a BPIV3 genomicpromoter, a gene encoding a BPIV3 N protein, the heterologous geneencoding the recombinant RSV F ectodomain, a gene encoding a BPIV3 Pprotein, a gene encoding a BPIV3 M protein, a gene encoding a HPIV3 Fprotein, a gene encoding a HPIV3 HN protein, and a gene encoding and aBPIV3 L protein; and the RSV F ectodomain is linked to the TM and CT ofa BPIV3 F protein.
 17. The recombinant paramyxovirus of claim 16,wherein the BPIV3 N protein, the recombinant RSV F ectodomain linked tothe TM and CT from a BPIV3 F protein, the BPIV3 P protein, the BPIV3 Mprotein, the HPIV3 F protein, the HPIV3 HN protein, and the BPIV3 Lprotein comprise amino acid sequences at least 90% identical to SEQ IDNOs: 47, 21, 48, 49, 43, 101, and 52, respectively; and the HPIV3 HNprotein further comprises I263T and T370P substitutions.
 18. Therecombinant paramyxovirus of claim 17, wherein the BPIV3 N protein, therecombinant RSV F ectodomain linked to the TM and CT from a BPIV3 Fprotein, the BPIV3 P protein, the BPIV3 M protein, the HPIV3 F protein,the HPIV3 HN protein, and the BPIV3 L protein comprise amino acidsequences set forth as SEQ ID NOs: 47, 21, 48, 49, 43, 101, and 52,respectively; and the HPIV3 HN protein further comprises I263T and T370Psubstitutions.
 19. The recombinant paramyxovirus of claim 1, wherein therecombinant RSV F ectodomain linked to the TM and CT of theparainfluenza virus protein is encoded by the nucleic acid sequence setforth as SEQ ID NO:
 23. 20. A recombinant paramyxovirus, comprising: aviral genome comprising genes encoding parainfluenza virus N, P, M, F,HN, and L proteins, and further comprising a heterologous gene encodinga type I membrane protein comprising a recombinant respiratory syncytialvirus (RSV) F ectodomain linked to a transmembrane domain (TM) andcytoplasmic tail (CT) of a parainfluenza virus F protein; wherein therecombinant paramyxovirus is a recombinant human/bovine parainfluenzavirus 3 (B/HPIV3); and the recombinant RSV F ectodomain linked to the TMand CT from a BPIV3 F protein is encoded by the nucleic acid sequenceset forth as SEQ ID NO:
 23. 21. The recombinant paramyxovirus of claim20, wherein the viral genome comprises, from upstream to downstream, aBPIV3 genomic promoter, a gene encoding a BPIV3 N protein, theheterologous gene encoding the recombinant RSV F ectodomain, a geneencoding a BPIV3 P protein, a gene encoding a BPIV3 M protein, a geneencoding a HPIV3 F protein, a gene encoding a HPIV3 HN protein, and agene encoding a BPIV3 L protein.
 22. The recombinant paramyxovirus ofclaim 21, wherein the BPIV3 N protein, the BPIV3 P protein, the BPIV3 Mprotein, the HPIV3 F protein, the HPIV3 HN protein, and the BPIV3 Lprotein comprise amino acid sequences at least 90% identical to SEQ IDNOs: 47, 21, 48, 49, 43, 101, and 52, respectively; and the HPIV3 HNprotein further comprises I263T and T370P substitutions.
 23. Therecombinant paramyxovirus of claim 22, wherein the BPIV3 N protein, theBPIV3 P protein, the BPIV3 M protein, the HPIV3 F protein, the HPIV3 HNprotein, and the BPIV3 L protein comprise amino acid sequences set forthas SEQ ID NOs: 47, 21, 48, 49, 43, 101, and 52, respectively; and theHPIV3 HN protein further comprises the I263T and T370P substitutions.24. The recombinant paramyxovirus of claim 20, wherein the heterologousgene encoding the recombinant RSV F ectodomain is the first or secondgene downstream of a genomic promoter of the viral genome.
 25. Therecombinant paramyxovirus of claim 20, wherein at least 90% of viralparticles produced by a host cell infected with the recombinantparamyxovirus or viral vector comprise a viral envelope comprising theectodomain encoded by the heterologous gene.
 26. The recombinantparamyxovirus of claim 20, wherein the recombinant paramyxovirus is aninfectious, attenuated, and self-replicating virus.
 27. The recombinantparamyxovirus of claim 20, wherein the RSV F ectodomain is present onthe viral envelope of the paramyxovirus.
 28. An immunogenic compositioncomprising the recombinant paramyxovirus of claim 20 and apharmaceutically acceptable carrier.
 29. A method of eliciting an immuneresponse to respiratory syncytial virus and parainfluenza virus in asubject comprising administering a therapeutically effective amount ofthe immunogenic composition of claim 28 to the subject.
 30. A nucleicacid molecule comprising the genome of the recombinant paramyxovirus ofclaim 20.